Methods for therapeutic administration of messenger ribonucleic acid drugs

ABSTRACT

The disclosure features methods of reducing or inhibiting an anti-drug antibody response in a subject, as well as methods of reducing or inhibiting unwanted immune cell activation in a subject to be treated with a messenger RNA (mRNA), comprising administering to the subject a mRNA, e.g., a chemically modified messenger RNA (mmRNA), encoding a polypeptide of interest, wherein the mRNA comprises at least one microRNA (miR) binding site for a miR expressed in immune cells, such as miR-126 binding site and/or miR-142 binding site, such that an anti-drug antibody response to the polypeptide or interest, or unwanted immune cell activation (e.g., B cell activation, cytokine secretion), is reduced or inhibited in the subject. The disclosure further provides therapeutic treatment regimens designed to reduce or inhibit ADA or unwanted immune cell activation (e.g., B cell activation, cytokine secretion) in a subject being treated with mRNA-based therapeutics.

RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/761,220, filed on Mar. 19, 2018, which is a 35 U.S.C. 371 nationalstage filing of International Application No. PCT/US2016/055582, filedon Oct. 5, 2016, which claims priority to U.S. Provisional PatentApplication Ser. No. 62/237,462 filed Oct. 5, 2015; U.S. ProvisionalPatent Application Ser. No. 62/317,268 filed Apr. 1, 2016; U.S.Provisional Patent Application Ser. No. 62/317,271 filed Apr. 1, 2016;U.S. Provisional Patent Application Ser. No. 62/317,366 filed Apr. 1,2016; U.S. Provisional Patent Application Ser. No. 62/338,385 filed May18, 2016; U.S. Provisional Patent Application Ser. No. 62/338,386 filedMay 18, 2016; U.S. Provisional Patent Application Ser. No. 62/338,388filed May 18, 2016; and U.S. Provisional Patent Application Ser. No.62/350,149 filed Jun. 14, 2016. The contents of the aforementionedapplications are hereby incorporated by reference in their entireties.

REFERENCE TO SQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format via EFS-Web, and is herebyincorporated by reference in its entirety. Said ASCII copy, created onSep. 29, 2020, is named MDN_005_011USCN_SL.txt and is 49379 bytes insize.

BACKGROUND OF THE INVENTION

Biologics, such as recombinant antibodies, cytokines and growth factors,have been shown to be effective in the treatment of a wide variety ofdiseases and the FDA has now approved a large number of such agents foruse in humans (for a review, see Kinch, M. S. (2015) Drug Discov. Today20:393-398). The vast majority of FDA approved biologics areprotein-based agents. More recently, messenger RNA-based agents arebeing developed as a disruptive therapeutic modality. There are severalreported examples of effective mRNA-based vaccines including bothinfectious disease vaccines and tumor vaccines (for respective reviews,see Marć M. A., et al. (2015) Expert Opin Drug Deliv. September 12:1-15and Sahin, U., et al. (2014) Nature Reviews Drug Discovery 13:759-780).The use of mRNA-based agents is more-recently being pursued fortherapeutic purposes, for example, using mRNA constructs that encode atherapeutic protein of interest.

Accordingly, new approaches and methods for use of mRNA-based agents ina subject, such as mRNA-based therapeutic agents, are needed,particularly methods that offer advantageous properties with regard tothe safety and/or therapeutic efficacy of the mRNA-based agent in thesubject.

SUMMARY OF THE INVENTION

The invention provides methods for use with mRNA-based agents to beadministered to a subject, wherein the methods provide advantageousfeatures for use of the mRNA-based agents in vivo. It has now beensurprisingly discovered that administration to non-human primates ofmRNA-based agents encoding a protein of interest can lead to developmentof an undesirable immune response in animals, wherein antibodies againstthe protein encoded by the mRNA can be detected in the animal. This isan unexpected result, since the animal was not administered a proteintherapeutic, but rather an mRNA construct, and it was not expected thatlocal production of the protein of interest in target tissues in vivowould lead to a response against the encoded protein product. Theresponse observed in non-human primates has also been studied in otherrelevant animal model systems and is analogous to the art-recognizedanti-drug antibody (ADA) response seen in both the fields of recombinantprotein therapeutics and even small molecule therapeutics. Arecognizable distinction in terminology is apparent to the skilledartisan in that a classic anti-drug antibody (ADA) response is generallyunderstood to be in response to systemic administration of, for example,a recombinant protein therapeutic, which can generate antibodiesdirectly to said protein therapeutic. In the field of mRNA therapeutics,the antibody responses observed in the herein-described animal studiesare not to the mRNA-based drug per se. By contrast, the antibodyresponses are to the mRNA drug-encoded protein product. The skilledartisan can refer to such a phenomenon as anti-protein antibody (APA)but owing to the pharmacologically analogous effects, the instantapplication will utilize the art-recognized terminology of anti-drugantibody (ADA).

Remarkably, it has now also been discovered that inclusion in the mRNAconstruct of at least one microRNA (miR) binding site for a miRexpressed in conventional immune cells or any cell that expresses TLR7and/or TLR8 and secretes pro-inflammatory cytokines and/or chemokines(e.g., in immune cells of peripheral lymphoid organs and/or splenocytesand/or endothelial cells) results in dramatic inhibition of ADAresponses in vivo. Accordingly, the disclosure provides methods forreducing or inhibiting anti-drug antibody responses in which a proteinof interest is encoded by a messenger RNA (mRNA) that comprises at leastone microRNA (miR) binding site for a miR expressed in conventionalimmune cells or any cell that expresses TLR7 and/or TLR8 and secretespro-inflammatory cytokines and/or chemokines (e.g., in immune cells ofperipheral lymphoid organs, such as spleen cells, e.g., splenic myeloidcells, and/or endothelial cells). In exemplary aspects, the disclosureprovides methods for reducing or inhibiting anti-drug antibody responsesin which a protein of interest is encoded by a messenger RNA (mRNA),e.g., modified messenger RNA (mmRNA), that comprises one or moremodified nucleobases and the mRNA, e.g., mmRNA, further comprises atleast one microRNA (miR) binding site for a miR expressed inconventional immune cells or any cell that expresses TLR7 and/or TLR8and secretes pro-inflammatory cytokines and/or chemokines (e.g., inimmune cells of peripheral lymphoid organs, such as spleen cells, e.g.,splenic myeloid cells, and/or endothelial cells). In the methods of thedisclosure, a subject is administered the mRNA, e.g., mmRNA, encodingthe polypeptide of interest and comprising the binding site for the atleast one immune cell-expressed miR such that an anti-drug antibodyresponse to the polypeptide of interest is reduced or inhibited in thesubject. In exemplary embodiments, the miR binding site is for a miRexpressed abundantly or preferentially in immune cells (e.g., in immunecells of peripheral lymphoid organs and/or splenocytes). In exemplaryembodiments, the miR binding site is included in an untranslated region(UTR) of the mRNA, e.g., mmRNA, encoding the protein of interest (e.g.,the 3′ UTR, the 5′ UTR, or in both the 3′ and 5′ UTRs). Thus, in themethods of the disclosure, an anti-drug antibody response is reduced orinhibited by post-transcriptional regulation of mRNA, with a possiblecomponent from translational repression, by inclusion of at least onemiR binding site, without the need to alter the amino acid sequence ofthe protein of interest.

In one embodiment, the at least one miR expressed in immune cells is amiR-142-3p microRNA binding site. In another embodiment, the at leastone miR expressed in immune cells is a miR-126 microRNA binding site,such as a miR-126-3p binding site. Accordingly, the disclosure providesa method of reducing or inhibiting an anti-drug antibody response in asubject, comprising administering to the subject a messenger RNA (mRNA),e.g., a modified messenger RNA (mmRNA), encoding a polypeptide ofinterest, wherein the mRNA, e.g., mmRNA, comprises at least onemiR-142-3p microRNA binding site and/or at least one miR-126 microRNAbinding site, and wherein the mRNA, e.g., mmRNA, comprises one or moremodified nucleobases, such that an anti-drug antibody response to thepolypeptide of interest is reduced or inhibited in the subject. In oneembodiment, the miR-142-3p microRNA binding site comprises the sequenceshown in SEQ ID NO: 3. In one embodiment, the miR-126 binding site is amiR-126-3p binding site. In one embodiment, the miR-126-3p microRNAbinding site comprises the sequence shown in SEQ ID NO: 26.

In other embodiments, the mRNA, e.g., mmRNA, comprises at least onemicroRNA binding site for a miR selected from the group consisting ofmiR-142, miR-146 miR-155, miR-126, miR-16, miR-21, miR-223, miR-24 andmiR-27. A miR referred to by number herein can refer to either of thetwo mature microRNAs originating from opposite arms of the samepre-miRNA (e.g., either the 3p or 5p microRNA). All miRs referred to bynumber herein are intended to include both the 3p and 5p arms/sequences.

In another embodiment, the mRNA, e.g., mmRNA, comprises at least two miRbinding sites for microRNAs expressed in immune cells. In variousembodiments, the mRNA, e.g., mmRNA, comprises 1-4, one, two, three orfour miR binding sites for microRNAs expressed in immune cells. ThesemiR binding sites can be for microRNAs selected from the groupconsisting of miR-142 (including miR-142-3p and miR-142-5p), miR-146(including miR-146-3p and miR-146-5p), miR-155, miR-126 (includingmiR-126-3p and miR-126-5p), miR-16, miR-21, miR-223, miR-24, miR-27, andcombinations thereof. In another embodiment, the mRNA, e.g., mmRNA,comprises at least two miR binding sites for microRNAs expressed inimmune cells, wherein one of the miR binding sites is for miR-142-3p. Invarious embodiments, the mRNA, e.g., mmRNA, comprises binding sites formiR-142-3p and miR-155, miR-142-3p and miR-146, or miR-142-3p andmiR-126 (e.g., miR-126-3p). In another embodiment, the mRNA, e.g.,mmRNA, comprises at least two miR binding sites for microRNAs expressedin immune cells, wherein one of the miR binding sites is for miR-126(e.g, miR-126-3p). In various embodiments, the mRNA, e.g., mmRNA,comprises binding sites for miR-126 and miR-155, miR-126 and miR-146, ormiR-126 and miR-142. In one embodiment, the mRNA, e.g., mmRNA, comprisesa miR-142-3p binding site and a miR-126 binding site.

In one embodiment, the mRNA, e.g., mmRNA, comprises a 5′ UTR, a codonoptimized open reading frame encoding the polypeptide of interest, a 3′UTR comprising the at least one microRNA binding site for a miRexpressed in immune cells, and a 3′ tailing region of linkednucleosides. In various embodiments, the 3′ UTR comprises 1-4, at leastone, two, three or four microRNA binding sites for miRs expressed inimmune cells, preferably abundantly or preferentially expressed inimmune cells (e.g., in immune cells of peripheral lymphoid organs and/orsplenocytes). In other embodiments, the 3′ UTR comprises at least onemiR-142-3p microRNA binding site or at least two miR binding sites formiRs expressed in immune cells, wherein one miR binding site is formiR-142-3p. In another embodiment, the 3′ UTR comprises at least onemiR-126 microRNA binding site or at least two miR binding sites for miRsexpressed in immune cells, wherein one miR binding site is for miR-126.In one embodiment, the codon optimized open reading frame encoding thepolypeptide of interest comprises a stop codon and the at least onemicroRNA binding site (e.g., a miR-142-3p binding site and/or a miR-126binding site) is located within the 3′ UTR 1-100 nucleotides after thestop codon. In another embodiment, the codon optimized open readingframe encoding the polypeptide of interest comprises a stop codon andthe at least one microRNA binding site (e.g., a miR-142-3p binding siteand/or a miR-126 binding site) is located within the 3′ UTR 30-50nucleotides after the stop codon. In another embodiment, the codonoptimized open reading frame encoding the polypeptide of interestcomprises a stop codon and the at least one microRNA binding site (e.g.,a miR-142-3p microRNA binding site and/or a miR-126 microRNA bindingsite) is located within the 3′ UTR at least 50 nucleotides after thestop codon.

In another embodiment, the mRNA, e.g., mmRNA, comprises a 5′ UTR and3′UTR which are heterologous to the coding region.

In another embodiment, the chemically modified mRNA, e.g., mmRNA, isfully modified. In other embodiments, the chemically modified mRNA,e.g., mmRNA, comprises one or more modified nucleobases describedfurther herein.

In some embodiments, the mRNA comprises pseudouridine (ψ). In someembodiments, the mRNA comprises pseudouridine (ψ) and 5-methyl-cytidine(m⁵C). In some embodiments, the mRNA comprises 1-methyl-pseudouridine(m¹ψ). In some embodiments, the mRNA comprises 1-methyl-pseudouridine(m¹ψ) and 5-methyl-cytidine (m⁵C). In some embodiments, the mRNAcomprises 2-thiouridine (s²U). In some embodiments, the mRNA comprises2-thiouridine and 5-methyl-cytidine (m⁵C). In some embodiments, the mRNAcomprises 5-methoxy-uridine (mo⁵U). In some embodiments, the mRNAcomprises 5-methoxy-uridine (mo⁵U) and 5-methyl-cytidine (m⁵C). In someembodiments, the mRNA comprises 2′-O-methyl uridine. In someembodiments, the mRNA comprises 2′-O-methyl uridine and5-methyl-cytidine (m⁵C). In some embodiments, the mRNA comprisescomprises N6-methyl-adenosine (m⁶A). In some embodiments, the mRNAcomprises N6-methyl-adenosine (m⁶A) and 5-methyl-cytidine (m⁵C).

In some embodiments, the modified nucleobase is pseudouridine (ψ),N1-methylpseudouridine (m¹ψ), 2-thiouridine, 4′-thiouridine,5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methoxyuridine, or 2′-O-methyl uridine. In some embodiments, an mRNAof the disclosure includes a combination of one or more of theaforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 ofthe aforementioned modified nucleobases.)

In some embodiments, the modified nucleobase is 1-methyl-pseudouridine(m¹ψ), 5-methoxy-uridine (mo⁵U), 5-methyl-cytidine (m⁵C), pseudouridine(ψ), α-thio-guanosine, or α-thio-adenosine. In some embodiments, an mRNAof the disclosure includes a combination of one or more of theaforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 ofthe aforementioned modified nucleobases.)

In one embodiment, the mRNA, e.g., mmRNA, is administered intravenouslyencapsulated in a lipid nanoparticle. In one embodiment, the lipidnanoparticle is a liposome. In one embodiment, the lipid nanoparticlecomprises a cationic and/or ionizable lipid. In one embodiment, thecationic and/or ionizable lipid is DLin-KC2-DMA or DLin-MC3-DMA.

In one embodiment, the polypeptide of interest is a therapeutic protein,a cytokine, a growth factor, an antibody or a fusion protein. Furtherexamples of polypeptides of interest are described herein.

In one embodiment, the mRNA, e.g., mmRNA, is administered by once weeklyinfusion. In another embodiment, the infusion is intravenously. Inanother embodiment, the mRNA, e.g., mmRNA, is administered by onceweekly infusion for at least 4 weeks. In another embodiment, the mRNA,e.g., mmRNA, is administered intratumorally. Suitable dosage regimensare described further herein.

In another embodiment, the disclosure provides a method of reducing orinhibiting an anti-drug antibody response following repeatedadministration of a polypeptide of interest to a subject, comprisingadministering to the subject intravenously a first dose of a mRNA, e.g.,modified mRNA (mmRNA), encoding a polypeptide of interest encapsulatedin an LNP, wherein the mRNA, e.g., mmRNA, comprises at least onemiR-142-3p microRNA binding site and/or at least one miR-126 microRNAbinding site, and wherein the mRNA, e.g., mmRNA, comprises one or moremodified nucleobases; and administering to the subject intravenously asecond dose of the mRNA, e.g., mmRNA, encapsulated in an LNP, such thatan anti-drug antibody response to the polypeptide of interest is reducedor inhibited in the subject.

In another aspect, the disclosure provides a method of reducing orinhibiting an anti-drug antibody response following repeatedadministration of a polypeptide of interest to a subject, comprising

(i) administering to the subject intravenously a first dose of a mRNA,e.g., modified mRNA (mmRNA), encoding a polypeptide of interestencapsulated in an LNP, wherein the mRNA, e.g., mmRNA, comprises atleast one microRNA binding site for a miR expressed in immune cells(e.g., a miR-142-3p microRNA binding site and/or a miR-126 microRNAbinding site), and wherein the mRNA, e.g., mmRNA, comprises one or moremodified nucleobases;

(ii) detecting a level of anti-drug antibodies in a sample from thesubject; and

(iii) administering to the subject intravenously a second dose of themRNA, e.g., mmRNA, encapsulated in an LNP when the level of anti-drugantibodies in the sample is diminished, such that an anti-drug antibodyresponse to the polypeptide of interest is reduced or inhibited in thesubject.

In another aspect, the disclosure provides a method of reducing orinhibiting drug-related toxicity in a subject, comprising administeringto the subject a messenger RNA (mRNA), e.g., a modified messenger RNA(mmRNA), encoding a polypeptide of interest, wherein the mRNA, e.g.,mmRNA, comprises at least one microRNA binding site for a miR expressedin immune cells (e.g., a miR-142-3p microRNA binding site and/or amiR-126 microRNA binding site), and wherein the mRNA, e.g., mmRNA,comprises one or more modified nucleobases, such that drug-relatedtoxicity to the polypeptide of interest is reduced or inhibited in thesubject. In one embodiment, the drug-related toxicity to the polypeptideof interest is decreased blood cell counts (cytopenia) in the subject.In one embodiment, the drug-related toxicity to the polypeptide ofinterest is autoimmunity in the subject. In one embodiment, thedrug-related toxicity to the polypeptide of interest is complementmediated effects in the subject. In one embodiment, the drug-relatedtoxicity to the polypeptide of interest is decreased hematopoiesis inthe subject. In other embodiments, the drug-related toxicity can be, forexample, renal toxicity or liver toxicity.

Additionally, it has now further been discovered that inclusion of atleast one binding site for a microRNA (miR) expressed in conventionalimmune cells or any cell that expresses TLR7 and/or TLR8 and secretespro-inflammatory cytokines and/or chemokines (e.g., in immune cells ofperipheral lymphoid organs, such as spleen cells, e.g., splenic myeloidcells, and/or endothelial cells), such as a miR-126 and/or miR-142binding site(s), in an mRNA reduces or inhibits unwanted immune cellactivation (e.g., B cell activation, cytokine secretion) in a subject towhom the mRNA is administered. It has been further discovered thatinclusion of this at least one miR binding site(s) in an mRNA can reduceor inhibit accelerated blood clearance (ABC) of a lipid-comprisingcompound or composition in which the mRNA is administered. Moreover,inclusion of this at least on miR binding site(s) in an mRNA can reduceor inhibit proliferation and/or activation of plasmacytoid dendriticcells (pDCs) and/or reduce or inhibit production of IgMs against thelipid-comprising compound or composition in which the mRNA isadministered by B cells, such as, for example, IgMs againstphsospholipid components (e.g., phosphatidylcholine) of thelipid-comprising compound or composition by B cells.

Accordingly, in one aspect, the disclosure provides methods for reducingor inhibiting unwanted immune cell activation in a subject administeredan RNA, e.g., mRNA encoding a polypeptide of interest, the methodscomprising administering to the subject an RNA, e.g., mRNA, e.g., achemically modified messenger RNA (mmRNA), encoding a polypeptide ofinterest, which comprises at least one binding site for a microRNA (miR)expressed in conventional immune cells or any cell that expresses TLR7and/or TLR8 and secretes pro-inflammatory cytokines and/or chemokines(e.g., in immune cells of peripheral lymphoid organs, such as spleencells, e.g., splenic myeloid cells, and/or endothelial cells), such as amiR-126 and/or miR-142 microRNA binding site, such that unwanted immunecell activation is reduced or inhibited in the subject. In anotheraspect, the disclosure provides methods for reducing or inhibitingunwanted cytokine production in a subject administered an RNA, e.g.,mRNA encoding a polypeptide of interest, the methods comprisingadministering to the subject an RNA, e.g., mRNA, e.g., a chemicallymodified messenger RNA (mmRNA), encoding a polypeptide of interest,which comprises at least one binding site for a microRNA (miR) expressedin conventional immune cells or any cell that expresses TLR7 and/or TLR8and secretes pro-inflammatory cytokines and/or chemokines (e.g., inimmune cells of peripheral lymphoid organs, such as spleen cells, e.g.,splenic myeloid cells, and/or endothelial cells), such as a miR-126and/or miR-142 microRNA binding site, such that unwanted cytokineproduction is reduced or inhibited in the subject. The RNA can be anmRNA, such as a chemically modified mRNA (referred to herein as anmmRNA) that comprises one or more modified nucleobases. The mmRNA can befully modified (i.e., all nucleotides or nucleobases of a particulartype are modified within the mmRNA), can be partially modified (i.e., aportion of nucleotides or nucleobases of a particular type are modifiedwithin the mRNA or can be a chimeric mRNA containing stretches ofmodified and unmodified nucleobases.

In one embodiment, reduction or inhibition of unwanted immune cellactivation and/or cytokine production is determined compared toadministration of a control RNA, e.g., mRNA, e.g., mmRNA, lacking the atleast one binding site for a microRNA (miR) expressed in immune cells,such as a miR-126 and/or miR-142 microRNA binding site. In oneembodiment, immune cell activation is decreased by at least 10%. Inanother embodiment, immune cell activation is decreased by at least 25%.In yet another embodiment, immune cell activation is decreased by atleast 50%. In still another embodiment, immune cell activation isdecreased without a corresponding decrease in expression of apolypeptide (e.g., therapeutic protein) of interest encoded by the mRNA.

In one embodiment, the immune cell activation is lymphocyte activation.In one embodiment, the lymphocyte activation is B cell activation. Inone embodiment, B cell activation is determined by frequency of CD19⁺CD86⁺ CD69⁺ B cells. In another embodiment, B cell activation isdetermined by cytokine secretion, e.g., in the serum or by total spleniccells. In one embodiment, B cell activation is determined by secretionof interleukin-6 (IL-6), tumor necrosis factor α (TNF-α) or interferon-γ(IFN-γ), e.g., in the serum or by total splenic cells. In oneembodiment, B cell activation is determined by secretion of IL-6, e.g.,in the serum or by total splenic cells. In one embodiment, reduction orinhibition of cytokine production is determined by reduction orinhibition of interleukin-6 (IL-6), tumor necrosis factor α (TNF-α) orinterferon-γ (IFN-γ) production. In another embodiment, reduction orinhibition of cytokine production is determined by reduction orinhibition of interleukin-6 (IL-6) production.

In another embodiment, the disclosure provides a method of reducing orinhibiting accelerated blood clearance in a subject repeatedlyadministered a messenger RNA (mRNA) encoding a polypeptide of interestencapsulated in an lipid nanoparticle (LNP), the method comprisingadministering to the subject a chemically modified mRNA encoding thepolypeptide of interest encapsulated in an lipid nanoparticle (LNP),wherein the chemically modified mRNA comprises at least one microRNAbinding site for a microRNA expressed in immune cells, and wherein thechemically modified mRNA comprises one or more modified nucleobases,such that accelerated blood clearance is reduced or inhibited in thesubject upon repeat administration.

In some embodiments, the disclosure provides a method of reducing orinhibiting accelerated blood clearance in a subject administered amessenger RNA (mRNA) encoding a polypeptide of interest encapsulated inan lipid nanoparticle (LNP), comprising administering to the subjectintravenously a first dose of a chemically modified mRNA encapsulated inan lipid nanoparticle (LNP), wherein the chemically modified mRNAcomprises at least one microRNA binding site for a microRNA expressed inimmune cells, and wherein the chemically modified mRNA comprises one ormore modified nucleobases; and administering to the subjectintravenously a second dose of the chemically modified mRNA encapsulatedin an LNP, such that accelerated blood clearance is reduced or inhibitedin the subject.

In some embodiments, the disclosure provides a method of reducing orinhibiting production of IgM molecules that recognize polyethyleneglycol (PEG) in a subject repeatedly administered a messenger RNA (mRNA)encoding a polypeptide of interest encapsulated in an lipid nanoparticle(LNP), the method comprising administering to the subject a chemicallymodified mRNA encoding the polypeptide of interest encapsulated in anlipid nanoparticle (LNP), wherein the chemically modified mRNA comprisesat least one microRNA binding site for a microRNA expressed in immunecells, and wherein the chemically modified mRNA comprises one or moremodified nucleobases, such that production of IgM molecules thatrecognize PEG are reduced or inhibited in the subject upon repeatadministration.

In further embodiments, the disclosure provides a method of reducing orinhibiting activation of B1a cells in a subject repeatedly administereda messenger RNA (mRNA) encoding a polypeptide of interest encapsulatedin an lipid nanoparticle (LNP), the method comprising administering tothe subject a chemically modified mRNA encoding the polypeptide ofinterest encapsulated in an lipid nanoparticle (LNP), wherein thechemically modified mRNA comprises at least one microRNA binding sitefor a microRNA expressed in immune cells, and wherein the chemicallymodified mRNA comprises one or more modified nucleobases, such thatactivation of B1a cells is reduced or inhibited in the subject uponrepeat administration.

In some embodiments, the disclosure provides a method of reducing orinhibiting activation of plasmacytoid dendrtic cells in a subjectrepeatedly administered a messenger RNA (mRNA) encoding a polypeptide ofinterest encapsulated in an lipid nanoparticle (LNP), the methodcomprising administering to the subject a chemically modified mRNAencoding the polypeptide of interest encapsulated in an lipidnanoparticle (LNP), wherein the chemically modified mRNA comprises atleast one microRNA binding site for a microRNA expressed in immunecells, and wherein the chemically modified mRNA comprises one or moremodified nucleobases, such that activation of plasmacytoid dendriticcells is reduced or inhibited in the subject upon repeat administration.

In further embodiments, the LNP does not activate B cells and/or doesnot induce production of IgM molecules capable of binding to the LNP,such that accelerated blood clearance is reduced or inhibited in thesubject upon administration of one or more subsequent doses. In someembodiments, the IgM molecules recognize polyethylene glycol (PEG).

In some embodiments, the reduction or inhibition of accelerated bloodclearance is determined compared to control administration of achemically modified mRNA lacking the at least one microRNA binding siteencapsulated in a lipid nanoparticle (LNP). In further embodiments, theaccelerated blood clearance is reduced or inhibited without acorresponding reduction or inhibition in expression of the polypeptideof interest encoded by the chemically modified mRNA.

In some embodiments, the interval between two consecutive doses is lessthan 2 weeks. In other embodiments, the interval between two consecutivedoses is less than 1 week.

In one embodiment, the mRNA, e.g., mmRNA, comprises a 5′ UTR, a codonoptimized open reading frame encoding a polypeptide of interest, a 3′UTR comprising the at least one binding site for a microRNA (miR)expressed in immune cells, such as a miR-126 (e.g., miR-126-3p) ormiR-142 (e.g., miR-142-3p) microRNA binding site, and a 3′ tailingregion of linked nucleosides. In one embodiment, the codon optimizedopen reading frame encoding the polypeptide of interest comprises a stopcodon and the at least one binding site for a microRNA expressed inimmune cells (e.g., a miR-142-3p binding site and/or a miR-126-3pbinding site) is located within the 3′ UTR 1-100 nucleotides after thestop codon. In another embodiment, the codon optimized open readingframe encoding the polypeptide of interest comprises a stop codon andthe at least one binding site for a microRNA (miR) expressed in immunecells, such as a miR-126 and/or miR-142 microRNA binding site is locatedwithin the 3′ UTR 30-50 nucleotides after the stop codon. In anotherembodiment, the codon optimized open reading frame encoding thepolypeptide of interest comprises a stop codon and the at least onebinding site for a microRNA (miR) expressed in immune cells, such as amiR-126 and/or miR-142 microRNA binding site is located within the 3′UTR at least 50 nucleotides after the stop codon. In another embodiment,the codon optimized open reading frame encoding the polypeptide ofinterest comprises a stop codon and the at least one binding site for amicroRNA expressed in immune cells (e.g., a miR-142-3p binding siteand/or a miR-126-3p binding site) is located anywhere in the 3′UTR(e.g., after the first 100 nucleotides after the stop codon). In anotherembodiment, the mRNA, e.g., mmRNA, comprises a 5′ UTR and 3′UTR whichare heterologous to the open reading frame.

In various embodiments, the mRNA, e.g., mmRNA, comprises 1-4, one, two,three or four miR binding sites for microRNAs expressed in immune cells,wherein at least one of the miR binding sites is a miR-126 binding site.In one embodiment, the mRNA, e.g., mmRNA, comprises at least twomicroRNA binding sites for microRNAs expressed in immune cells, whereinat least one of the microRNA binding sites is a miR-126 binding site. Inone embodiment, the mRNA, e.g., mmRNA, comprises a miR-126 binding siteand a second microRNA binding site for a miR selected from the groupconsisting of miR-142-3p, miR-142-5p, miR-146-3p, miR-146-5p, miR-155,miR-16, miR-21, miR-223, miR-24 and miR-27. In another embodiment, themRNA, e.g., mmRNA comprises a miR-126 (e.g., miR-126-3p) binding siteand a miR-142 (e.g., miR-142-3p) binding site. In one embodiment, themRNA, e.g., mmRNA, comprises at least three microRNA binding sites formicroRNAs expressed in immune cells, wherein at least one of themicroRNA binding sites is a miR-126 binding site. In one embodiment, themRNA, e.g., mmRNA, comprises a miR-126 binding site, a miR-142 (e.g.,miR_142-3p) binding site, and a third microRNA binding site for a miRselected from the group consisting of miR-146-3p, miR-146-5p, miR-155,miR-16, miR-21, miR-223, miR-24 and miR-27. In another embodiment, themRNA, e.g., mmRNA, comprises a miR-126 binding site, a miR-142 (e.g.,miR-142-3p) binding site, and a miR-155 binding site. In one embodiment,the mRNA, e.g., mmRNA, comprises at least four microRNA binding sitesfor microRNAs expressed in immune cells. In another embodiment, themRNA, e.g., mmRNA, comprises a miR-126 binding site, a miR-142-3pbinding site, a miR-142-5p binding site, and a miR-155 binding site. AmiR referred to by number herein can refer to either of the two maturemicroRNAs originating from opposite arms of the same pre-miRNA (e.g.,either the 3p or 5p microRNA). All miRs referred to by number herein areintended to include both the 3p and 5p arms/sequences.

In one embodiment, the miR-126-3p binding site comprises the sequenceshown in SEQ ID NO: 26.

In one embodiment, the miR-142-3p binding site comprises the sequenceshown in SEQ ID NO: 3.

In one embodiment, the miR-155 binding site comprises the sequence shownin SEQ ID NO: 35.

In some embodiments, the microRNA binding site binds a microRNAexpressed in myeloid cells. In other embodiments, the microRNA bindingsite binds a microRNA expressed in plasmacytoid dendritic cells. In yetother embodiments, the microRNA binding site binds a microRNA expressedin macrophages.

In another embodiment, the mRNA, e.g., mmRNA, is fully modified for aparticular chemical modification. Types of suitable chemicalmodification are described further herein. In other embodiments, themRNA, e.g., mmRNA, comprises one or more modified nucleotides ornucleobases described further herein.

In some embodiments, the mRNA comprises pseudouridine (ψ). In someembodiments, the mRNA comprises pseudouridine (ψ) and 5-methyl-cytidine(m⁵C). In some embodiments, the mRNA comprises 1-methyl-pseudouridine(m¹ψ). In some embodiments, the mRNA comprises 1-methyl-pseudouridine(m¹ψ) and 5-methyl-cytidine (m⁵C). In some embodiments, the mRNAcomprises 2-thiouridine (s²U). In some embodiments, the mRNA comprises2-thiouridine and 5-methyl-cytidine (m⁵C). In some embodiments, the mRNAcomprises 5-methoxy-uridine (mo⁵U). In some embodiments, the mRNAcomprises 5-methoxy-uridine (mo⁵U) and 5-methyl-cytidine (m⁵C). In someembodiments, the mRNA comprises 2′-O-methyl uridine. In someembodiments, the mRNA comprises 2′-O-methyl uridine and5-methyl-cytidine (m⁵C). In some embodiments, the mRNA comprisescomprises N6-methyl-adenosine (m⁶A). In some embodiments, the mRNAcomprises N6-methyl-adenosine (m⁶A) and 5-methyl-cytidine (m⁵C).

In some embodiments, the modified nucleobase is pseudouridine (ψ),N1-methylpseudouridine (m¹ψ), 2-thiouridine, 4′-thiouridine,5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methoxyuridine, or 2′-O-methyl uridine. In some embodiments, an mRNAof the disclosure includes a combination of one or more of theaforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 ofthe aforementioned modified nucleobases.)

In some embodiments, the modified nucleobase is 1-methyl-pseudouridine(m¹ψ), 5-methoxy-uridine (mo⁵U), 5-methyl-cytidine (m⁵C), pseudouridine(ψ), α-thio-guanosine, or α-thio-adenosine. In some embodiments, an mRNAof the disclosure includes a combination of one or more of theaforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 ofthe aforementioned modified nucleobases.)

In one embodiment, the mRNA, e.g., mmRNA, is administered intravenouslyencapsulated in a lipid nanoparticle. In one embodiment, the lipidnanoparticle is a liposome. In one embodiment, the lipid nanoparticlecomprises a cationic and/or ionizable lipid. In one embodiment, thecationic and/or ionizable lipid is DLin-KC2-DMA or DLin-MC3-DMA.

In one embodiment, the mRNA, e.g., mmRNA, encodes a polypeptide ofinterest. In various embodiments, the polypeptide of interest is atherapeutic protein, a cytokine, a growth factor, an antibody or afusion protein. Further examples of polypeptides of interest aredescribed herein.

In one embodiment, the mRNA, e.g., mmRNA, is administered by once weeklyinfusion. In another embodiment, the infusion is intravenously. Inanother embodiment, the mRNA, e.g., mmRNA, is administered by onceweekly infusion for at least 4 weeks. In another embodiment, the mRNA,e.g., mmRNA, is administered intratumorally. Suitable dosage regimensare described further herein.

In another embodiment, the disclosure provides a method of reducing orinhibiting unwanted immune cell activation (e.g., B cell activation)and/or unwanted cytokine production in a subject administered amessenger RNA (mRNA) encoding a polypeptide of interest, the methodcomprising administering to the subject intravenously a first dose of amRNA, e.g., modified mRNA (mmRNA) encoding a polypeptide of interestencapsulated in an LNP, wherein the mRNA, e.g., mmRNA, comprises atleast one binding site for a microRNA (miR) expressed in immune cells,such as a miR-126 microRNA binding site and/or at least one miR-142microRNA binding site,

and wherein the mRNA, e.g., mmRNA, comprises one or more modifiednucleobases; and administering to the subject intravenously a seconddose of the mRNA, e.g., mmRNA, encapsulated in an LNP, such thatunwanted immune cell activation and/or unwanted cytokine production isreduced or inhibited in the subject.

In another aspect, the disclosure provides a method of reducing orinhibiting unwanted immune cell activation (e.g., B cell activation)and/or unwanted cytokine production in subject following repeatedadministration of a messenger RNA (mRNA) encoding a polypeptide ofinterest to a subject, comprising

(i) administering to the subject intravenously a first dose of a mRNA,e.g., modified mRNA (mmRNA) encoding a polypeptide of interest,encapsulated in an LNP, wherein the mRNA, e.g., mmRNA, comprises atleast one binding site for a microRNA (miR) expressed in immune cells,such as a miR-126 microRNA binding site and/or at least one miR-142microRNA binding site, and wherein the mRNA, e.g., mmRNA, comprises oneor more modified nucleobases;

(ii) detecting a level of immune cell activation in a sample from thesubject; and

(iii) administering to the subject intravenously a second dose of themRNA, e.g., mmRNA, encapsulated in an LNP when the level of immune cellactivation in the sample is diminished, such that unwanted immune cellactivation and/or unwanted cytokine production is reduced or inhibitedin the subject.

In yet another aspect, the disclosure provides a modified messenger RNA(mmRNA) encoding a polypeptide of interest, wherein the mmRNA comprisesat least two different microRNA (miR) binding sites, wherein themicroRNA is expressed in an immune cell of hematopoietic lineage or acell that expresses TLR7 and/or TLR8 and secretes pro-inflammatorycytokines and/or chemokines, and wherein the mmRNA comprises one or moremodified nucleobases. In some aspects, the immune cell of hematopoieticlineage is a lymphoid cell, such as a T cell, B cell, or NK cell. Insome aspects, the immune cell of hematopoietic lineage is a myeloidcell, such as a monocyte, macrophage, neutrophil, basophil, eosinophil,erthyrocyte, dendritic cell, megakaryocyte, or platelet. In someaspects, the immune cell of hematopoietic lineage is a hematopoieticprogenitor cell. In some aspects, the cell that expresses TLR7 and/orTLR8 and secretes pro-inflammatory cytokines and/or chemokines is anendothelial cell.

In some aspects of the disclosure, the mmRNA comprises at least twodifferent microRNA binding sites, wherein the microRNA is abundant inthe same or different cell type of interest. In some aspects themicroRNA is abundant in multiple cell types of interest.

In some aspects, the disclosure provides an mmRNA comprising at leastone first microRNA binding site of a microRNA abundant in an immune cellof hematopoietic lineage and at least one second microRNA binding siteof a microRNA abundant in endothelial cells, wherein the mmRNA comprisesone or more modified nucleobases.

In some aspects, the disclosure provides an mmRNA comprising at leastone first microRNA binding site of a microRNA abundant in B cells and atleast one second microRNA binding site of a microRNA abundant inendothelial cells, wherein the mmRNA comprises one or more modifiednucleobases.

In some aspects, the disclosure provides an mmRNA comprising at leastone first microRNA binding site of a microRNA abundant in plasmacytoiddendritic cells and at least one second microRNA binding site of amicroRNA abundant in endothelial cells, wherein the mmRNA comprises oneor more modified nucleobases.

In some aspects of the disclosure, the mmRNA comprises multiple copies(2, 3, 4 copies) of a first microRNA binding site and at least one copyof a second microRNA binding site. In some aspects, the mmRNA comprises2 copies of a first microRNA binding site and 1 copy of a secondmicroRNA binding site.

In some aspects, the disclosure provides an mmRNA comprising first andsecond microRNA binding sites of the same microRNA, such as, forexample, microRNA binding sites of the 3p and 5p arms of the samemicroRNA.

Some aspects of the disclosure provide a modified messenger RNA (mmRNA)encoding a polypeptide of interest, wherein the mmRNA comprises at leasttwo different microRNA (miR) binding sites, wherein the microRNA isselected from the group consisting of miR-126, miR-142, miR-144,miR-146, miR-150, miR-155, miR-16, miR-21, miR-223, miR-24, miR-27 andmiR-26a, and wherein the mmRNA comprises one or more modifiednucleobases. In some aspects, the microRNA is selected from the groupconsisting of miR126-3p, miR-142-3p, miR-142-5p, and miR-155. In someaspects, the at least one microRNA binding site is a miR-126 bindingsite, such as set forth in SEQ ID NO: 26. In some aspects, the at leastone microRNA binding site is a miR-142 binding site, such as set forthin SEQ ID NO: 3.

In yet other aspects, the disclosure provide a modified messenger RNA(mmRNA) encoding a polypeptide of interest, wherein the mmRNA comprisesat least two different microRNA (miR) binding sites, wherein onemicroRNA binding site is a miR-126 binding site and the second microRNAbinding site is for a microRNA selected from the group consisting ofmiR-142-3p, miR-142-5p, miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21,miR-223, miR-24 and miR-27, and wherein the mmRNA comprises one or moremodified nucleobases.

In other aspects, the disclosure provide a modified messenger RNA(mmRNA) encoding a polypeptide of interest, wherein the mmRNA amiR-126-3p binding site and a miR-142-3p binding site, and wherein themmRNA comprises one or more modified nucleobases. In some aspects themmRNA comprises in the 5′ or 3′ UTR a single miR-126-3p binding site asset forth in SEQ ID NO: 26 and a single miR-142-3p binding site as setforth in SEQ ID NO: 3. In some aspects the mmRNA comprises at least onemiR-142-3p binding site and at least one 142-5p binding site, such asset forth in SEQ ID NO: 3 and SEQ ID NO: 51, respectively.

In yet other aspects, the disclosure provide a modified messenger RNA(mmRNA) encoding a polypeptide of interest, wherein the mmRNA comprisesat least three different microRNA binding sites, wherein at least one ofthe microRNA binding sites is a miR-126 binding site, and wherein themmRNA comprises one or more modified nucleobases.

In some aspects, the disclosure provide a modified messenger RNA (mmRNA)encoding a polypeptide of interest, wherein the mmRNA comprises at leastthree different microRNA binding sites, wherein at least one of themicroRNA binding sites is a miR-142 binding site, and wherein the mmRNAcomprises one or more modified nucleobases.

In yet other aspects, the disclosure provide a modified messenger RNA(mmRNA) encoding a polypeptide of interest, wherein the mmRNA comprisesat least one miR-126-3p binding site, at least one miR-142-3p, and athird microRNA binding site for a microRNA selected from the groupconsisting of miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223,miR-24 and miR-27, and wherein the mmRNA comprises one or more modifiednucleobases. In some aspects, the mmRNA comprises at least onemiR-126-3p binding site, at least one miR-142-3p binding site, and atleast one miR-155 binding site (e.g., a 155-5p binding site as set forthin the Sequence Listing). In some aspects, the mmRNA comprises at leastone miR-126-3p binding site, at least one miR-142-3p binding site, atleast one miR-142-5p binding site, and at least one miR-155 bindingsite.

In any of the preceding and related aspects, the disclosure provides anmmRNA comprising a 5′ UTR, a codon optimized open reading frame encodingthe polypeptide of interest, a 3′ UTR, and a 3′ tailing region of linkednucleosides, wherein the microRNA binding sites are located in the 5′UTR, 3′ UTR, or both the 5′ UTR and 3′ UTR of the mmRNA. In someaspects, the microRNA binding sites are located in the 3′ UTR of themmRNA. In some aspects, the microRNA binding sites are located in the 5′UTR of the mmRNA. In some aspects, the microRNA binding sites arelocated in both the 5′ UTR and 3′ UTR of the mmRNA. In some aspects, theat least one microRNA binding site is located in the 3′ UTR immediatelyadjacent to the stop codon of the coding region of the mmRNA. In someaspects, the at least one microRNA binding site is located in the 3′ UTR70-80 bases downstream of the stop codon of the coding region of themmRNA. In some aspects, the at least one microRNA binding site islocated in the 5′ UTR immediately preceding the start codon of thecoding region of the mmRNA. In some aspects, the at least one microRNAbinding site is located in the 5′ UTR 15-20 nucleotides preceding thestart codon of the coding region of the mmRNA. In some aspects, the atleast one microRNA binding site is located in the 5′ UTR 70-80nucleotides preceding the start codon of the coding region of the mmRNA.

In some aspects, the disclosure provides mmRNA comprising multiplecopies of the same or different microRNA binding sites positionedimmediately adjacent to each other or with a spacer of less than 5,5-10, 10-15, or 15-20 nucleotides, in either the 5′ UTR, 3′ UTR or both.In some aspects, the mmRNA comprises multiple copies of the samemicroRNA binding site located in the 3′ UTR, wherein the first microRNAbinding site is positioned immediately adjacent to the stop codon andthe second and third microRNA binding sites are positioned 30-40 basesdownstream of the first microRNA binding site. In some aspects, themmRNA comprises 2 copies of a first microRNA binding site and 1 copy ofa second microRNA binding site located in the 3′ UTR, wherein the firstcopy of the first microRNA binding site is positioned immediatelyadjacent to the stop codon, the second microRNA binding site ispositioned 30-40 bases downstream of the first copy of the firstmicroRNA binding site, and the second copy of the first microRNA bindingsite is positioned 30-40 bases downstream of the second microRNA bindingsite.

In any of the foregoing or related aspects, the disclosure provides amodified mRNA wherein the mmRNA is fully modified.

In any of the foregoing or related aspects, the disclosure provides anmmRNA comprising pseudouridine (ψ), pseudouridine (ψ) and5-methyl-cytidine (m⁵C), 1-methyl-pseudouridine (m¹ψ),1-methyl-pseudouridine (m¹ψ) and 5-methyl-cytidine (m⁵C), 2-thiouridine(s²U), 2-thiouridine and 5-methyl-cytidine (m⁵C), 5-methoxy-uridine(mo⁵U), 5-methoxy-uridine (mo⁵U) and 5-methyl-cytidine (m⁵C),2′-O-methyl uridine, 2′-O-methyl uridine and 5-methyl-cytidine (m⁵C),N6-methyl-adenosine (m⁶A) or N6-methyl-adenosine (m⁶A) and5-methyl-cytidine (m⁵C).

In any of the foregoing or related aspects, the disclosure provides anmmRNA comprising pseudouridine (ψ), N1-methylpseudouridine (m¹ψ),2-thiouridine, 4′-thiouridine, 5-methylcytosine,2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine,2-thio-5-aza-uridine, 2-thio-dihydropseudouridine,2-thio-dihydrouridine, 2-thio-pseudouridine,4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine,4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine,dihydropseudouridine, 5-methoxyuridine, or 2′-O-methyl uridine, orcombinations thereof.

In any of the foregoing or related aspects, the disclosure provides anmmRNA comprising 1-methyl-pseudouridine (m¹ψ), 5-methoxy-uridine (mo⁵U),5-methyl-cytidine (m⁵C), pseudouridine (ψ), α-thio-guanosine, orα-thio-adenosine, or combinations thereof.

In any of the foregoing or related aspects, the disclosure provides anmmRNA encoding a polypeptide of interest, wherein the polypeptide ofinterest is a therapeutic protein, cytokine, growth factor, antibody orfusion protein.

In any of the foregoing or related aspects, the disclosure provides alipid nanoparticle comprising a modified mRNA as described herein. Insome aspects, the lipid nanoparticle comprises a liposome. In someaspects, the lipid nanoparticle comprises a cationic and/or ionizablelipid. In some aspects, the cationic and/or ionizable lipid isDLin-KC2-DMA or DLin-MC3-DMA.

In any of the foregoing or related aspects, the disclosure provides apharmaceutical composition comprising the mmRNA or lipid nanoparticle asdescribed herein, and a pharmaceutically acceptable carrier, diluent orexcipient.

In any of the foregoing or related aspects, the disclosure provides anmmRNA, a lipid nanoparticle or a pharmaceutical composition as describedherein, for use in reducing or inhibiting an anti-drug antibody responseor inhibiting drug-related toxicity in a subject in need thereof.

In any of the foregoing or related aspects, the disclosure provides anmmRNA, a lipid nanoparticle or a pharmaceutical composition as describedherein, for use in reducing or inhibiting unwanted immune cellactivation or reducing or inhibiting unwanted cytokine production in asubject in need thereof.

In any of the foregoing or related aspects, the disclosure provides anmmRNA, a lipid nanoparticle or a pharmaceutical composition as describedherein, for use in reducing or inhibiting accelerated blood clearance ina subject in need thereof.

In any of the foregoing or related aspects, the disclosure provides anmmRNA, a lipid nanoparticle or a pharmaceutical composition as describedherein, for use in reducing or inhibiting production of IgM moleculesthat recognize polyethylene glycol (PEG) in a subject in need thereof.

The details of various embodiments of the disclosure are set forth inthe description below. Other features, objects, and advantages of thedisclosure will be apparent from the description and the drawings, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the levels of human erythropoietin (hEPO)protein in cynomolgus macaques treated with mmRNA encoding hEPO (butlacking any miR binding sites) at the indicated doses, or withrecombinant hEPO protein as a positive control, six hours post infusionon the indicated days of treatment.

FIG. 2 is a schematic diagram of mRNA constructs without and with aninserted miR site(s) in the 3′ UTR.

FIG. 3 is a graph comparing the anti-hEPO antibody levels in cynomolgusmacaques treated with PBS or with 0.2 mg/kg of an mmRNA encoding hEPO,either lacking or containing a miR-142-3p binding site in the 3′ UTR ofthe construct. Animals positive for an anti-drug antibody (ADA) responseare indicated.

FIGS. 4A-C are graphs showing protein expression levels (FIG. 4A), Bcell frequency (FIG. 4B) and activated B cell frequency (FIG. 4C) inmice treated with 0.05 mg/kg mmRNA encoding hEPO either lacking orcontaining a miR-142-3p binding site, a miR-126 binding site, or boththe miR-142-3p and miR-126 binding sites, in the 3′ UTR of theconstruct.

FIGS. 5A-B are graphs showing protein expression levels for mice treatedwith a single dose of 0.2 mg/kg (FIG. 5A) or 1 mg/kg (FIG. 5B) of mmRNAencoding hEPO either lacking or containing a miR-142-3p binding site, amiR-126 binding site, or both the miR-142-3p and miR-126 binding sites,in the 3′ UTR of the construct.

FIGS. 6A-B are graphs showing protein expression levels for mice treatedwith two doses of 0.2 mg/kg (FIG. 6A) or 1 mg/kg (FIG. 6B) of mmRNAencoding hEPO either lacking or containing a miR-142-3p binding site, amiR-126 binding site, or both the miR-142-3p and miR-126 binding sites,in the 3′ UTR of the construct.

FIG. 7 are graphs showing B cell frequency of mice treated with a singleindicated dose (0.2 mg/kg or 1 mg/kg) of mmRNA encoding hEPO eitherlacking or containing a miR-142-3p binding site, a miR-126 binding site,or both the miR-142-3p and miR-126 binding sites, in the 3′ UTR of theconstruct.

FIG. 8 are graphs showing activated B cell frequency of mice treatedwith a single indicated dose (0.2 mg/kg or 1 mg/kg) of mmRNA encodinghEPO either lacking or containing a miR-142-3p binding site, a miR-126binding site, or both the miR-142-3p and miR-126 binding sites, in the3′ UTR of the construct.

FIG. 9 are graphs showing B cell frequency of mice treated with twodoses of the indicated dosage (0.2 mg/kg or 1 mg/kg) of mmRNA encodinghEPO either lacking or containing a miR-142-3p binding site, a miR-126binding site, or both the miR-142-3p and miR-126 binding sites, in the3′ UTR of the construct.

FIG. 10 are graphs showing activated B cell frequency of mice treatedwith two doses of the indicated dosage (0.2 mg/kg or 1 mg/kg) of mmRNAencoding hEPO either lacking or containing a miR-142-3p binding site, amiR-126 binding site, or both the miR-142-3p and miR-126 binding sites,in the 3′ UTR of the construct.

FIGS. 11A-B are graphs showing IL-6 levels in mice treated with a singledose of 0.2 mg/kg (FIG. 11A) or 1 mg/kg (FIG. 11B) of mmRNA encodinghEPO either lacking or containing a miR-142-3p binding site, a miR-126binding site, or both the miR-142-3p and miR-126 binding sites, in the3′ UTR of the construct.

FIGS. 12A-B are graphs showing IL-6 levels in mice treated with twodoses of 0.2 mg/kg (FIG. 12A) or 1 mg/kg (FIG. 12B) of mmRNA encodinghEPO either lacking or containing a miR-142-3p binding site, a miR-126binding site, or both the miR-142-3p and miR-126 binding sites, in the3′ UTR of the construct.

FIGS. 13A-C are graphs showing IL-6 levels (FIG. 13A), TNF-α levels(FIG. 13B) and IFN-γ levels (FIG. 13C) in mice treated with two doses of0.2 mg/kg mmRNA encoding hEPO either lacking or containing a miR-142-3pbinding site, a miR-126 binding site, or both the miR-142-3p and miR-126binding sites, in the 3′ UTR of the construct.

FIGS. 14A-C are graphs showing IL-6 levels (FIG. 14A), TNF-α levels(FIG. 14B) and IFN-γ levels (FIG. 14C) in mice treated with two doses of1 mg/kg mmRNA encoding hEPO either lacking or containing a miR-142-3pbinding site, a miR-126 binding site, or both the miR-142-3p and miR-126binding sites, in the 3′ UTR of the construct.

FIGS. 15A-B are graphs showing Luciferase (Luc) expression levels, asmeasured by whole body luminescence, in mice treated for 1 week (FIG.15A) or two weeks (FIG. 15B) with 0.2 mg/kg mmRNA encoding Luc eitherlacking or containing a miR-142-3p binding site, a miR-126 binding site,or both the miR-142-3p and miR-126 binding sites, in the 3′ UTR of theconstruct.

FIG. 16 are graphs showing total B cell frequency in mice treated with a0.2 mg/kg mmRNA encoding Luc either lacking or containing a miR-142-3pbinding site, a miR-126 binding site, or both the miR-142-3p and miR-126binding sites, in the 3′ UTR of the construct.

FIG. 17 are graphs showing activated B cell frequency in mice treatedwith 0.2 mg/kg mmRNA encoding Luc either lacking or containing amiR-142-3p binding site, a miR-126 binding site, or both the miR-142-3pand miR-126 binding sites, in the 3′ UTR of the construct.

FIGS. 18A-C are graphs showing secreted IL-6 levels (FIG. 18A), TNF-αlevels (FIG. 18B) and IFN-γ levels (FIG. 18C) in mice treated with 0.2mg/kg mmRNA encoding Luc either lacking or containing a miR-142-3pbinding site, a miR-126 binding site, or both the miR-142-3p and miR-126binding sites, in the 3′ UTR of the construct.

FIG. 19A-B are graphs showing EPO expression levels in the serum of micetreated for 1 week (FIG. 19A) or two weeks (FIG. 19B) with 0.2 mg/kgmmRNA encoding EPO either lacking or containing a miR-142-3p bindingsite, a miR-142-5p binding site, a miR-155-5p binding site, or multiplecopies or combinations thereof.

FIG. 20 are graphs showing total B cell frequency in mice treated for 1week with 0.2 mg/kg mmRNA encoding EPO either lacking or containing amiR-142-3p binding site, a miR-142-5p binding site, a miR-155-5p bindingsite, or multiple copies or combinations thereof.

FIG. 21 are graphs showing activated B cell frequency in mice treatedfor 1 week with 0.2 mg/kg mmRNA encoding EPO either lacking orcontaining a miR-142-3p binding site, a miR-142-5p binding site, amiR-155-5p binding site, or multiple copies or combinations thereof.

FIG. 22 are graphs showing total B cell frequency in mice treated fortwo weeks with 0.2 mg/kg mmRNA encoding EPO either lacking or containinga miR-142-3p binding site, a miR-142-5p binding site, a miR-155-5pbinding site, or multiple copies or combinations thereof.

FIG. 23 are graphs showing activated B cell frequency in mice treatedfor 2 weeks with 0.2 mg/kg mmRNA encoding EPO either lacking orcontaining a miR-142-3p binding site, a miR-142-5p binding site, amiR-155-5p binding site, or multiple copies or combinations thereof.

FIGS. 24A-C are graphs showing secreted IL-6 levels (FIG. 24A), TNF-αlevels (FIG. 24B) and IFN-γ levels (FIG. 24C) in mice treated for 2weeks with 0.2 mg/kg mmRNA encoding EPO either lacking or containing amiR-142-3p binding site, a miR-142-5p binding site, a miR-155-5p bindingsite, or multiple copies or combinations thereof.

FIGS. 25A-B are graphs showing the percentage of CD27⁺ CD19⁺ B cells insplenic CD19⁺ B cells (FIG. 25A) and the level of CD27 expression inCD27⁺ CD19⁺ B cells (FIG. 25B) in mice treated with mmRNA encoding EPOeither lacking or containing a miR-142 binding site, a miR-126 bindingsite or miR-142 and miR-126 binding sites.

FIGS. 26A-B are graphs showing the total CD11c⁺ cell frequency insplenic cells (FIG. 26A) and the percentage of activated dendritic cells(CD11 CD70⁺ CD86⁺ cells) (FIG. 26B) in mice treated with mmRNA encodingEPO either lacking or containing a miR-142 binding site, a miR-126binding site or miR-142 and miR-126 binding sites.

FIG. 27 is a graph showing the level of proliferation of naïve B cellsin the presence of plasmacytoid dendritic cells (pDCs) isolated frommice treated with mmRNA encoding EPO either lacking or containing amiR-142 binding site, a miR-126 binding site or miR-142 and miR-126binding sites.

FIGS. 28A-C are graphs showing the level of serum anti-PEG IgMantibodies in mice treated with two doses (FIG. 28A), three doses (FIG.28B) or four doses (FIG. 28C) of mmRNA encoding EPO either lacking orcontaining a miR-142 binding site, a miR-126 binding site or miR-142 andmiR-126 binding sites.

FIG. 29 is a graph showing EPO expression levels in the serum of micetreated for 6 weeks with 0.2 mg/kg mmRNA encoding EPO either lacking orcontaining a miR-142-3p binding site, a miR-126 binding site, or boththe miR-142-3p and miR-126 binding sites, in the 3′ UTR of theconstruct.

FIG. 30 is a graph showing Luciferase (Luc) expression levels in theserum of mice treated for 5 weeks with 0.2 mg/kg mmRNA encoding Luceither lacking or containing a miR-142-3p binding site, a miR-126binding site, or both the miR-142-3p and miR-126 binding sites, in the3′ UTR of the construct.

FIGS. 31A-D are graphs showing the level of expression of eGFP inprimary hepatocytes transfected with an equimolar mixture of Luc andeGFP mRNA constructs in LNP, wherein the mRNA constructs containedeither no recognizable miR sites (control), 1× or 3×miR-122 bindingsites or a putative mRNA with similar sequence to the eGFP and Lucsequences (control), at doses of 7.5 ng (FIG. 31A), 15 ng (FIG. 31B), 50ng (FIG. 31C) or 100 ng (FIG. 31D).

FIGS. 32A-D are graphs showing the percent phase confluence (as ameasure of caspase-mediated toxicity) of primary hepatocytes transfectedwith caspase mRNA constructs containing either no recognizable miR sites(control), 1× or 3×miR-122 binding sites or a putative mRNA with acaspase-like sequence (control) with no start codon, at doses of 7.5 ng(FIG. 32A), 15 ng (FIG. 32B), 50 ng (FIG. 32C) or 100 ng (FIG. 32D).

FIG. 33 is a graph showing the level of expression of hEPO incynomologus monkeys administered mRNA constructs containing either norecognizable miR sites (control), 1× or 3×miR-142-3p binding sites.

FIG. 34 is a bar graph showing the level of expression of eGFP inRAW264.7 cells transfected with mRNA constructs containing either norecognizable miR sites (control), a miR-142-3p binding site in the 3′UTR (1× or 3×), a miR-142-3p binding site in the 5′ UTR (inserted at P1,P2 or P3), or a miR-142-3p binding site in both the 3′ UTR and the 5′UTR.

FIG. 35 is a bar graph showing the level of expression of hEPO inprimary hepatocytes cells transfected with mRNA constructs containingeither no recognizable miR sites (control), a miR-122 binding site inthe 3′ UTR (1× or 3×), a miR-122 binding site in the 5′ UTR (inserted atP1, P2 or P3), or a miR-122 binding site in both the 3′ UTR and the 5′UTR.

DETAILED DESCRIPTION

One challenge associated with the clinical use of protein-basedtherapeutics is the development of an unwanted anti-drug antibody (ADA)response, wherein the patient's immune system generates antibodiesagainst the therapeutic agent (for reviews, see e.g., Subramanyam, M.(2006) J. Immunotoxicol. 3:151-156; De Groot, A. S. and Scott, D. W.(2007) Trends Immunol. 28:482-490; Nechansky, A. and Kircheis, R. (2010)Expert Opin. Drug. Discov. 5:1067-1079). Development of ADA responseshas been reported both for recombinant antibody biologics and fornon-antibody biologics (see e.g., Brickelmaier, M. et al. (1999) JImmunol. Methods 227:121-135; Ruf, P. et al. (2010) Br. J. Clin.Pharmacol. 69:617-625; Lundkvist, M. et al. (2013) Mult. Scler.19:757-764). The ADA response can interfere with or neutralize theeffect of the therapeutic agent, thereby impacting drug pharmacokineticsand efficacy. Neutralizing antibodies (NAB) are generally of moreconcern than binding antibodies (BAB) that are not neutralizing, butboth may have clinical consequences.

Furthermore, allergic reactions, complement activation and other adverseevents are often associated with the development of ADA, therebyimpacting drug safety. Thus, ADA is a significant factor in the abilityto use biologics for long-term treatment.

The use of modified mRNA, e.g., mRNAs (mmRNAs), as therapeutic agentsoffers an exciting alternative to protein-based therapeutics. mRNAtherapeutics offer several advantages over the protein-based therapeuticart, including, for example, fidelity of encoded protein characteristics(because the protein is produced by the body's own translationapparatus), sensitive, tunable pharmacokinetic profile (proteinexpression may be transient, which may be favorable for some therapeuticapproaches to better control pharmacokinetics and dosing), excellentsafety profile (as revealed in various vaccine clinical trials),functionality in the cytoplasm without the need to travel to the nucleusresulting in protein translation almost immediately after mRNAadministration, eliminates any risk of genomic integration, as well asease of manufacturing, e.g., mRNAs are easily produced by variousgeneric, in vitro processes, e.g., in vitro transcription reactions,without the need for living organisms. Furthermore, mRNA can be designedeither to have self-adjuvanting properties, e.g., in vaccineapplications, or to evade immunogenic activation, e.g., in therapeuticapplications. It has now been discovered, however, that administrationof mRNA encoding a protein of interest, particularly in instances wherethe mRNA administration leads directly or indirectly to expression ofthe encoded protein in immune cells, e.g., the spleen, also can lead tothe development of an anti-drug antibody response to the protein encodedby the mRNA. It has surprisingly been demonstrated, however, thatincorporation of at least one microRNA (miRNA) binding site for a miRexpressed in immune cells (e.g., in immune cells of peripheral lymphoidorgans and/or splenocytes) into the mRNA (mRNA) encoding the protein ofinterest can reduce the anti-drug antibody response to the protein ofinterest when the mRNA is administered to the subject.

Accordingly, the disclosure provides methods for reducing or inhibitingan anti-drug antibody (ADA) response to a protein of interest by meansof post-transcriptional regulation, in particular in immune systemtissue such as the spleen. The disclosure also provides methods ofreducing drug-related toxicity in a subject by incorporation of at leastone microRNA (miRNA) binding site for a miR expressed in immune cellsinto a mRNA (e.g., mmRNA) encoding a protein of interest. PreferredmicroRNA binding sites used in the methods of the disclosure are thosethat bind miRs expressed abundantly or preferentially in immune cells(e.g., in immune cells of peripheral lymphoid organs and/orsplenocytes). A particularly preferred microRNA binding site is formiR-142-3p. Another particularly preferred microRNA binding site is formiR-126.

As described in Example 1, an in vivo study in which cynomolgus macaqueswere administered an mmRNA construct encoding human erythropoietin(hEPO) led to the observations that the levels of hEPO declined overtime in the animals. Furthermore, reticulocytopenia and reduced bonemarrow hematopoiesis were also observed. These results suggested thepossibility that anti-drug antibodies were being generated in theanimals, which was confirmed by ELISA analysis of serum. While in no waybeing bound by theory, prior experience with the mRNA delivery systemused in the study (using lipid nanoparticles, LNPs) demonstrated thatthe mRNA distributed primarily to the liver but also to the spleen and,thus, this distribution could lead to heightened immunity to the proteinbeing made that is encoded by the mRNA. Again while not being bound bytheory or mechanism, it is possible that expression of encoded proteinmanufactured in the spleen (delivered based on the LNP distribution tothe spleen by direct delivery via the blood flow or indirectly viaprofessional antigen presenting cells) could lead to T cell dependentantibody production via the presentation of appropriate epitopes (i.e.,from the protein of interest) to T cells.

To address this, as described in Example 2, additional mRNA constructswere designed that included at least one microRNA (miRNA) binding sitefor a miR expressed in immune cells (e.g., at least one binding site formiR-142-3p). Administration of the miR-containing mRNA construct in vivoled to a significant reduction in the development of ADA responses inthe recipient animals.

Another challenge associated with the clinical use of protein-basedtherapeutics in the art is the development of unwanted immune cellactivation (e.g., B cell activation) against the therapeutic protein,leading to immune-mediated side effects. It has now been discovered,however, that administration of mRNA, e.g., encoding a protein ofinterest, particularly in instances where the mRNA administration leadsdirectly or indirectly to expression of the encoded protein in immunecells, e.g., splenocytes, also can lead to the development unwantedimmune cell activation (e.g., B cell activation, including cytokineproduction). It has surprisingly been demonstrated, however, thatincorporation of at least one binding site for a microRNA (miRNA) thatis expressed in peripheral lymphoid tissue and/or endothelial cells, inparticular at least one miR-126 and/or miR-142 binding site, into themRNA (mRNA) can reduce or inhibit unwanted immune cell activation whenthe mRNA is administered to the subject. Accordingly, the disclosureprovides compositions and methods for reducing or inhibiting unwantedimmune cell activation when using mRNA-based therapeutic agents by meansof post-transcriptional regulation, in particular in immune systemtissue such as peripheral lymphoid organs or the spleen.

Experiments described in Example 3 demonstrated that incorporation of amiR-126 or miR-142 (e.g., miR-142-3p) binding site, or the two sites incombination, into mRNA constructs encoding a protein of interest, led toa reduced frequency of activated B cells, as well as reduced levels ofcytokine production (IL-6, TNF-α, IFN-γ), in animals administered theconstructs, as compared to animals treated with constructs lacking themiR binding site(s). The effect of the miR-126 binding site alone wasmore potent than the effect of the miR-142 binding site alone, with thestrongest effects being seen with the two sites used in combination.Frequency of B cell activation and cytokine production are earlyindicators of a mounting immune response in vivo, including antibodyresponses. Thus, these results indicate that inclusion of a miR-126binding site in an mRNA construct (alone or in combination with amiR-142-3p binding site) can lead to a reduction in the development ofADA responses to the encoded protein in the recipient animal.

While in no way being bound by theory, the inclusion of a miR-126binding site in an mRNA construct can lead to reduced or inhibitedimmune cell activation by one or more possible mechanisms, based on theexpression pattern of miR-126. MicroRNA-126 is known to be highly andselectively expressed in plasmacytoid dendritic cells (pDCs) andregulates the maturation, survival and effector functions of these cells(Agudo, J. et al. (2014) Nat. Immunol. 15:54-62; Cella, M. andTrinchieri, G. (2014) Nat. Immunol. 15:8-9). Plasmacytoid dendriticcells account for less than 0.1% of peripheral blood mononuclear cellsand 0.4-0.6% of total splenic cells, can differentiate into dendriticcells upon activation, produce interferons and serve as a link betweeninnate and adaptive immunity, as well as playing a role in antigenpresentation (for reviews on pDCs, see e.g., Jegalian, A. G. et al.(2009) Adv. Anat. Pathol. 16:392-404; Reizis, B. et al. (2011) Annu.Rev. Immunol. 29:163-183; Tel, J. et al. (2012) Cancer Immunol.Immunotherap. 61:1279-1288). Furthermore, pDCs are also involved inpromoting B cell activation and differentiation and in stimulatingcytokine production (see e.g., Douag, I. et al. (2009) J. Immunol.182:1991-2001; Ding, C. et al. (2009) J. Immunol. 183:7140-7149; Gujer,C. et al. (2011) J. Leukoc. Biol. 89:811-821). Thus, the reducedfrequency of B cell activation and the reduced cytokine productionobserved by the inclusion of a miR-126 binding site in an mRNA constructmay result, for example, from inhibition of the antigen presentingfunction of the pDCs and/or from inability of pDCs to launch aneffective response against foreign nucleic acids and/or from inhibitionof the maturation and survival of the pDCs, thereby leading to reducedpromotion of B cell activation and reduced cytokine production, theoverall result of these effects then being a reduced ADA response invivo against the protein encoded by the mRNA construct.

Additionally, miR-126 is known to be expressed in endothelial cells (seee.g., Fish, J. E. et al. (2008) Dev. Cell. 15:272-284; Wang, S. et al.(2008) Dev. Cell. 15:-261-271). Accordingly, the effect of inclusion ofa miR-126 binding site in an mRNA construct may be related to theabundance of miR-126 in endothelial cells. Thus, inclusion of a miR-126binding site in an mRNA construct may lead to reduced expression of theencoded protein in endothelial cells in vivo, resulting in reducedantigen presentation by the endothelial cells, leading to a concomitantreduction in frequency of B cell activation and reduced cytokineproduction, resulting in reduced ADA responses against the encodedprotein in vivo.

As demonstrated in Example 6, inclusion of a miR-142 and/or miR-126binding site in an mRNA construct leads to reduced total frequency ofCD11c⁺ dendritic cells, as well as reduced frequency of activateddendritic cells (CD11c⁺ CD70⁺ CD86⁺ cells) within the CD11c⁺ spleniccell population. In contrast, inclusion of a miR-142 and/or miR-126binding site in mRNA constructs did not affect the frequency of CD27⁺CD19⁺ B cells in splenic CD19⁺ B cells, nor did it affect the level ofCD27 expression in the CD27⁺ CD19⁺ B cell population. Furthermore,proliferation of naïve B cells was reduced when incubated with pDCsisolated from mice treated with the miR binding site(s)-containingconstructs, as compared to treatment with an mRNA construct lacking themiR binding site(s). Thus, this experimental data supports the proposedmechanism that inhibition of B cell activation and inhibition ofcytokine production in mice treated with modified mRNA constructsincluding one or a combination of miR binding site(s) results fromdecreased frequency and/or activation of pDCs, thereby leading todecreased B cell stimulation, likely resulting from decreased CD70-CD27interactions or reduced dendritic cells cytokine secretion.

A separate challenge exists with the use of lipid-comprising compoundsand compositions, such as lipid nanoparticles (LNPs), to delivertherapeutic agents, e.g., modified mRNA, wherein the agents are rapidlycleared from the blood upon second and subsequent administrations (i.e.,accelerated blood clearance (ABC)). The mechanism includes therecognition of lipid-comprising compounds or compositions (e.g., LNPs)by B cells, in particular, by B1a cells, through CD36 and/or TLRrecognition of the lipid components, such as phosphatidylcholine.Activated B1a cells secrete IgM, in particular, natural IgM, which cancontribute to ABC (e.g., via an acute phase response-type mechanism.Phospholilid component (e.g., DSPC) of a lipid-comprising compound orcomposition (e.g., LNP) can also activate platelets, for example, incirculation. Activated platelets can aggregate and bind to macrophages,which subsequently release inflammatory cytokines and migrate to thespleen. The sequesteration of lipid-comprising compounds or compositions(e.g., LNPs) to the spleen happens almost immediately afteradministration.

It has been discovered that ABC is mediated, at least in part, by Bcells, specifically B1a cells. These B cells are normally responsiblefor secreting natural IgM antibodies, which are polyreactive, meaningthat they are able to bind to a variety of antigens, albeit withrelatively low affinity for each. Upon administration of a first dose ofan agent, B1a cells bind the agent and are activated, thereby secretingnatural IgM that binds to the agent, such as phosphatidylcholine. Asecond or subsequent dose of a lipid-comprising compound or compositionis then targetd by circulating IgM and rapidly cleared. Conventional Bcells, referred to herein as B2 cells or CD19(+) B cells, are alsoimplicated in ABC. Specifically, conventional B cells are able to mountfirst an IgM response followed by an IgG response concomitant with amemory response. The conventional B cells react against the administeredagent and the polyethylene glycol (PEG) and contribute to IgM (andeventually IgG) that mediates ABC. Previous solutions to this challengehave focused on supressing the immune response in subject administeredLNP compositions. In particular, co-medication regimens (e.g.,antihistamines, non-steroidal anti-inflammatory drugs (NSAIDs),steroids, corticosteroids, and the like) have been used to supress theimmune system. It has now been discovered, however, that incorporationof at least one microRNA binding site for a microRNA expressed in immunecells (e.g., miR-126, miR-142, miR-155 and combinations thereof) into amodified mRNA construct can reduce or inhibit ABC when thelipid-comprising compound or composition comprising the modified mRNA isadministered to a subject. Specifically, it has been discovered thatincorporation of at least one microRNA binding site into a modified mRNAcan reduce or inhibit plasmacytoid dendritic cell proliferation and/oractivation and/or reduce or inhibit production of anti-PEG IgMs. Forexample, as demonstrated in Example 7, inclusion of the at least one miRbinding site(s) in the mRNA construct leads to decreased levels of serumanti-PEG IgM antibodies in mice administered a lipid-comprising compoundor composition comprising the mRNA constructs.

Multiple possible mechanisms exist by which the inclusion of at leastone microRNA binding site(s), as described herein, into a modified mRNAconstruct being delivered by lipid-comprising compounds or compositionsleads to reduction or inhibition of ABC. In one embodiment, themechanism of action of the miRNA binding site(s) is a microRNA “sponge”,wherein the miRNA binding site(s) in the construct “soaks up” microRNAsthat bind to the binding site(s). This can lead to deregulation ofnatural targets of the specific microRNA as this microRNA is less/notavailable to regulate them. This scenario mimics the effects of amicroRNA knock-down/knock-out. In examples where proper regulation ofthe natural targets of the microRNA is necessary for the cell's abilityto act as an effective immune cell, this microRNA-spone-type effectrenders the cell incapable of producing an immune response. It is alsopossible that deregulation of an endogenous target of the microRNAdisrupts the homeostasis of the cell (e.g., calcium signaling), leadingto a stress response (e.g., unfolded protein response). Alternatively,it is possible that inclusion of the microRNA-binding in the mRNAsuppresses expression from this mRNA in the specific microRNA-harboringcell-type. It is also possible that inclusion of the microRNA bindingsite leads to degradation of the mRNA before a sensor like TLR7 canrecognize it. The latter two mechanisms are postulated to be dependenton RNA-induced silencing complex (RISC)-mediated cleavage of an mRNAcomprising a one or more binding sites for a microRNA (miR) expressed inimmune cells. It is also possible that these mechanisms act in concert,both leading to the miR-mediated observed effects described herein.

Regardless of the mechanisms involved, the resulting impact of theinclusion of at least one microRNA binding site(s), as described herein,into a mRNA construct is that immune cells which recognize thelipid-comprising compounds or compositions (e.g., pDCs, B cells (e.g.,circulating B cells, macrophages), are not activated and therefore donot migrate to the spleen to activate B cells (e.g, splenic B cells). Inaddition, cytokine production (e.g., IL-6) is reduced or inhibited whichfurther prevents activation of the immune cells. The reduction orinhibition of B cell activation results in a reduction or inhibition ofnatural IgMs (e.g., by B1a cells), IgMs and IgGs. The production ofthese molecules are essential for ABC and therefore the reduction orinhibition of their production reduces or inhibits ABC overall.

Accordingly, the disclosure provides methods for reducing or inhibitingABC when using lipid-comprising compounds or compositions comprisingmodified mRNA encoding a polypeptide of interest.

Various aspects of the disclosure are described further in thesubsections below.

mRNA

The disclosure provides isolated RNAs, in particular mRNAs, e.g.,chemically modified mRNAs, that encode a polypeptide of interest andthat include at least one microRNA binding site (e.g., miR-126 and/ormiR-142 binding sites). In other embodiments, the disclosure providesRNAs, e.g., chemically modified RNAs, that include at least one microRNAbinding site (e.g., miR-126 and/or miR-142 binding sites), but that donot necessarily encode a polypeptide of interest. The latter RNAs alsomay lack other typical features of mRNAs (such as the mRNA featuresdescribed below), yet include the miR-126 and/or miR-142 bindingsite(s).

An RNA may be a naturally or non-naturally occurring RNA, e.g., mRNA. AnmRNA may include one or more modified nucleobases, nucleosides, ornucleotides, as described below, in which case it may be referred to asa “chemically modified mRNA”, also referred to herein as a “modifiedmRNA” or “mmRNA.” As described herein “nucleoside” is defined as acompound containing a sugar molecule (e.g., a pentose or ribose) orderivative thereof in combination with an organic base (e.g., a purineor pyrimidine) or a derivative thereof (also referred to herein as“nucleobase”). As described herein, “nucleotide” is defined as anucleoside including a phosphate group.

An mRNA may include a 5′ untranslated region (5′UTR), a 3′ untranslatedregion (3′UTR), and/or a coding region (e.g., an open reading frame). AnmRNA may include any suitable number of base pairs, including hundreds(e.g., 200, 300, 400, 500, 600, 700, 800, or 900) or thousands (e.g.,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000) of basepairs. Any number (e.g., all, some, or none) of nucleobases,nucleosides, or nucleotides may be an analog of a canonical species,substituted, modified, or otherwise non-naturally occurring. In certainembodiments, all of a particular nucleotide or nucleobase type may bemodified.

In one embodiment, the mRNA comprises a first flanking region located atthe 5′ terminus of an open reading frame (coding region) and a secondflanking region located at the 3′ terminus of the open reading frame(coding region), wherein the first flanking region comprises a 5′untranslated region (5′ UTR) and the second flanking region comprises a3′ untranslated region (3′UTR). In one embodiment, the 5′UTR and the3′UTR of the mRNA are not derived from the same species. In oneembodiment, the 5′UTR and/or the 3′UTR of the mRNA are not derived frombeta-globin. In one embodiment, the 5′ untranslated region isheterologous to the coding region of the mRNA. In another embodiment,the 3′ untranslated region is heterologous to the coding region of themRNA. In yet another embodiment, the 5′ untranslated region and the 3′untranslated region are heterologous to the coding region of the mRNA.In yet another embodiment, the mRNA comprises at least two stop codons.

The sequence of a non-limiting example of a 5′ UTR suitable for use inthe mRNA constructs is shown in SEQ ID NO: 53. The sequence of anon-limiting example of a 3′ UTR suitable for use in the mRNA constructsis shown in SEQ ID NO: 30. Other suitable 5′ and 3′ UTRs suitable foruse in the mRNA constructs are well known in the art. For example,suitable 5′ UTRs include those from the β-globin gene (see e.g., Karikoet al. (2008) Mol. Therap. 16:1833-40; U.S. Pat. Nos. 8,278,063,9,012,219), the α-globin gene (see e.g., U.S. Pat. No. 9,012,219), thehuman cytochrome b-245 α polypeptide gene (CYBA) (see e.g., Ferizi etal. (2015) Lab. Chip. 23:1456-1464), the hydroxysteroid (1713)dehydrogenase gene (HSD17B4) (see e.g., Thess et al. (2015) Mol. Therap.23:1456-1464; WO 2015/024667), the TOP gene (see e.g., WO/2015101414,WO2015/101415, WO2015/062738, WO2015/024667, WO2015/024667), theribosomal protein Large 32 (L32) gene (see e.g., WO2015/101414,WO2015/101415, WO2015/062738) and the ATP51 gene (see e.g.,WO2015/024667), as well as viral 5′ UTRs, including those from Tobaccoetch virus (TEV) (see e.g., Katalin et al. (2012) Mol. Therap.20:948-953; U.S. Pat. Nos. 8,278,063, 9,012,219), Venezuelan equineencephalitis virus (VEEV), (see e.g., Andries et al. (2015) J. ControlRelease 217:337-344) and the CMV immediate-early 1 (IE1) gene (see e.g.,US20140206753, WO2014/089486, WO2013/185069, WO2014/144196,WO2014/152659, WO2014/152940, WO2014/152774, WO2014/153052). Synthetic5′ UTRs have been described and are also suitable for use (see e.g.,Mandal and Rossi (2013) Nat. Protocol 5:68-82).

Additionally, for example, suitable 3′ UTRs include those from theβ-globin gene (see e.g., Kariko et al. (2008) Mol. Therap. 16:1833-40;U.S. Pat. Nos. 8,278,063; 9,012,219; WO2007/036366, US 2011/0065103,WO2011/015347, WO2012/072096, WO2013/143555, WO2014/071963), theα-globin gene (see e.g., U.S. Pat. No. 9,012,219; WO2015/101414,WO2015/101415, WO2015024667), the human cytochrome b-245 α polypeptidegene (CYBA) (see e.g., Ferizi et al. (2015) Lab. Chip. 23:1456-1464),the albumin gene (see e.g., Thess et al. (2015) Mol. Therap.23:1456-1464), the human growth hormone (hGH) gene (see e.g.,US20140206753, WO2013/185069, WO2014/089486, WO2014/144196,WO2014/152659, WO2014152940, WO2014/152774, WO2014/153052), theribosomal rps9 protein gene (see e.g., WO2015/101414), the FIG. 4 gene(see e.g., WO2015/101415), the human albumin? gene (see e.g.,WO2015/101415, WO2015/101414, WO2015/06273, WO2015/024667,WO2105/062737), as well as viral 3′ UTRs, including those fromVenezuelan equine encephalitis virus (VEEV), (see e.g., Andries et al.(2015) J. Control Release 217:337-344).

In some embodiments, an mRNA as described herein may include a 5′ capstructure, a chain terminating nucleotide, a Kozak sequence (also knownas a Kozak consensus sequence), a stem loop, a polyA sequence, and/or apolyadenylation signal. In other embodiments, the mRNA lacks a poly Asequence and/or a polyadenylation signal but rather contains analternative structure for stabilizing the mRNA.

A 5′ cap structure or cap species is a compound including two nucleosidemoieties joined by a linker and may be selected from a naturallyoccurring cap, a non-naturally occurring cap or cap analog, or ananti-reverse cap analog (ARCA). A cap species may include one or moremodified nucleosides and/or linker moieties. For example, a natural mRNAcap may include a guanine nucleotide and a guanine (G) nucleotidemethylated at the 7 position joined by a triphosphate linkage at their5′ positions, e.g., m⁷G(5′)ppp(5′)G, commonly written as m⁷GpppG. A capspecies may also be an anti-reverse cap analog. A non-limiting list ofpossible cap species includes m⁷GpppG, m⁷Gpppm⁷G, m⁷3′dGpppG, m₂^(7,O3′)GpppG, m₂ ^(7,O3′)GppppG, m₂ ^(7,O2′)GppppG, m⁷Gpppm⁷G,m⁷3′dGpppG, m₂ ^(7,O3′)GpppG, m₂ ^(7,O3′)GppppG, and m₂ ^(7,O2′)GppppG.In various embodiments, the mRNA can comprise a 5′ terminal cap selectedfrom the group consisting of CapO, Capl, ARCA, inosine,N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine,8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and2-azido-guanosine. In one embodiment, the 5′ terminal cap is Capl.

An mRNA may instead or additionally include a chain terminatingnucleoside. For example, a chain terminating nucleoside may includethose nucleosides deoxygenated at the 2′ and/or 3′ positions of theirsugar group. Such species may include 3′-deoxyadenosine (cordycepin),3′-deoxyuridine, 3′-deoxycytosine, 3′-deoxyguanosine, 3′-deoxythymine,and 2′,3′-dideoxynucleosides, such as 2′,3′-dideoxyadenosine,2′,3′-dideoxyuridine, 2′,3′-dideoxycytosine, 2′,3′-dideoxyguanosine, and2′,3′-dideoxythymine. In some embodiments, incorporation of a chainterminating nucleotide into an mRNA, for example at the 3′-terminus mayresult in stabilization of the mRNA, as described, for example, inInternational Patent Publication No. WO 2013/103659.

An mRNA may instead or additionally include a stem loop, such as ahistone stem loop. A stem loop may include 2, 3, 4, 5, 6, 7, 8, or morenucleotide base pairs. For example, a stem loop may include 4, 5, 6, 7,or 8 nucleotide base pairs. A stem loop may be located in any region ofan mRNA. For example, a stem loop may be located in, before, or after anuntranslated region (a 5′ untranslated region or a 3′ untranslatedregion), a coding region, or a polyA sequence or tail. In someembodiments, a stem loop may affect one or more function(s) of an mRNA,such as initiation of translation, translation efficiency, and/ortranscriptional termination.

An mRNA may instead or additionally include a polyA sequence and/orpolyadenylation signal. A polyA sequence may be comprised entirely ormostly of adenine nucleotides or analogs or derivatives thereof. A polyAsequence may be a tail located adjacent to a 3′ untranslated region ofan mRNA. In some embodiments, a polyA sequence may affect the nuclearexport, translation, and/or stability of an mRNA.

In some embodiments, an mRNA is a bicistronic mRNA comprising a firstcoding region and a second coding region with an intervening sequencecomprising an internal ribosome entry site (IRES) sequence that allowsfor internal translation initiation between the first and second codingregions, or with an intervening sequence encoding a self-cleavingpeptide, such as a 2A peptide. IRES sequences and 2A peptides aretypically used to enhance expression of multiple proteins from the samevector. A variety of IRES sequences are known and available in the artand may be used, including, e.g., the encephalomyocarditis virus IRES.

In one embodiment, the polynucleotides of the present disclosure mayinclude a sequence encoding a self-cleaving peptide. The self-cleavingpeptide may be, but is not limited to, a 2A peptide. A variety of 2Apeptides are known and available in the art and may be used, includinge.g., the foot and mouth disease virus (FMDV) 2A peptide, the equinerhinitis A virus 2A peptide, the Thosea asigna virus 2A peptide, and theporcine teschovirus-1 2A peptide. 2A peptides are used by severalviruses to generate two proteins from one transcript byribosome-skipping, such that a normal peptide bond is impaired at the 2Apeptide sequence, resulting in two discontinuous proteins being producedfrom one translation event. As a non-limiting example, the 2A peptidemay have the protein sequence: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 4),fragments or variants thereof. In one embodiment, the 2A peptide cleavesbetween the last glycine and last proline. As another non-limitingexample, the polynucleotides of the present disclosure may include apolynucleotide sequence encoding the 2A peptide having the proteinsequence GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 4) fragments or variantsthereof. One example of a polynucleotide sequence encoding the 2Apeptide is: GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT (SEQ ID NO: 5). In one illustrative embodiment, a 2Apeptide is encoded by the following sequence:5′-TCCGGACTCAGATCCGGGGATCTCAAAATTGTCGCTCCTGTCAAACAAACTCTTAACTTTGATTTACTCAAACTGGCTGGGGATGTAGAAAGCAATCCAGGTCCACTC-3′(SEQ ID NO: 6).The polynucleotide sequence of the 2A peptide may be modified or codonoptimized by the methods described herein and/or are known in the art.

In one embodiment, this sequence may be used to separate the codingregions of two or more polypeptides of interest. As a non-limitingexample, the sequence encoding the 2A peptide may be between a firstcoding region A and a second coding region B (A-2Apep-B). The presenceof the 2A peptide results in the cleavage of one long protein intoprotein A, protein B and the 2A peptide. Protein A and protein B may bethe same or different peptides or polypeptides of interest.

Modified mRNAs

In some embodiments, an mRNA of the disclosure comprises one or moremodified nucleobases, nucleosides, or nucleotides (termed “chemicallymodified mRNAs”, also referred to herein as “modified mRNAs” or“mmRNAs”). In some embodiments, modified mRNAs may have usefulproperties, including enhanced stability, intracellular retention,enhanced translation, and/or the lack of a substantial induction of theinnate immune response of a cell into which the mRNA is introduced, ascompared to a reference unmodified mRNA. Therefore, use of modifiedmRNAs may enhance the efficiency of protein production, intracellularretention of nucleic acids, as well as possess reduced immunogenicity.

In some embodiments, an mRNA, includes one or more (e.g., 1, 2, 3 or 4)different modified nucleobases, nucleosides, or nucleotides. In someembodiments, an mRNA includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more) different modifiednucleobases, nucleosides, or nucleotides. In some embodiments, themodified mRNA, may have reduced degradation in a cell into which themmRNA is introduced, relative to a corresponding unmodified mRNA.

In one embodiment, the mRNA comprises at least one nucleoside (ornucleotide) modification. In another embodiment, the mRNA comprises atleast one modification as compared to the chemical structure of an A, G,U or C ribonucleoside. In yet another embodiment, the mRNA is anisolated polynucleotide comprising;

(a) a first region of linked nucleosides, said first region encoding apolypeptide of interest;

(b) a first flanking region located 5′ relative to said first regioncomprising a 5′ untranslated region (5′UTR) and at least one 5′ terminalcap;

(c) a second flanking region located 3′ relative to said first regioncomprising a 3′ untranslated region (3′UTR) and a 3′ tailing sequence oflinked nucleosides;

wherein said polynucleotide comprises at least one chemically modifiednucleoside.

In some embodiments, the modified nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having a modified uracil includepseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine,6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U),4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m³U),5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U),1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U),5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U),5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine(mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U),5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τm⁵U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(τm⁵s²U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U,i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m¹ψ,5-methyl-2-thio-uridine (m⁵ s²U), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ w),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um), and5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)]uridine.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine include5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine(m³C), N4-acetyl-cytidine (ac⁴C), 5-formyl-cytidine (f⁵C),N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C),1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine (s²C), 2-thio-5-methyl-cytidine,4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k2C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm),5,2′-O-dimethyl-cytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm),N4,2′-O-dimethyl-cytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine (f⁵Cm),N4,N4,2′-O-trimethyl-cytidine (m⁴ ₂Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine includeα-thio-adenosine, 2-amino-purine, 2, 6-diaminopurine,2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine(e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine,7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m′A),2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A),2-methylthio-N6-methyl-adenosine (ms² m⁶A), N6-isopentenyl-adenosine(i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A),N6-(cis-hydroxyisopentenyl)adenosine (io⁶A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms²io⁶A),N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine(t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A),2-methylthio-N6-threonylcarbamoyl-adenosine (ms²g⁶A),N6,N6-dimethyl-adenosine (m⁶ ₂A), N6-hydroxynorvalylcarbamoyl-adenosine(hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A),N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am),N6,2′-O-dimethyl-adenosine (m⁶Am), N6,N6,2′-O-trimethyl-adenosine (m⁶₂Am), 1,2′-0-dimethyl-adenosine (m¹Am), 2′-O-ribosyladenosine(phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine,8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine,2′-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeα-thio-guanosine, inosine (I), 1-methyl-inosine (m¹I), wyosine (imG),methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2),wybutosine (yW), peroxywybutosine (o₂yW), hydroxywybutosine (OhyW),undermodified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine(Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ),mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ₀),7-aminomethyl-7-deaza-guanosine (preQ₁), archaeosine (G⁺),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methyl-guanosine (m¹G), N2-methyl-guanosine (m²G),N2,N2-dimethyl-guanosine (m² ₂G), N2,7-dimethyl-guanosine (m^(2,7)G),N2, N2,7-dimethyl-guanosine (m^(2,2,7)G), (8-oxo-guanosine,7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine,α-thio-guanosine, 2′-O-methyl-guanosine (Gm),N2-methyl-2′-O-methyl-guanosine (m²Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m² ₂Gm),1-methyl-2′-O-methyl-guanosine (m¹Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m^(2,7)Gm), 2′-O-methyl-inosine(Im), 1,2′-O-dimethyl-inosine (m¹Im), 2′-O-ribosylguanosine (phosphate)(Gr(p)), 1-thio-guanosine, 06-methyl-guanosine, 2′-F-ara-guanosine, and2′-F-guanosine.

In some embodiments, an mmRNA, of the disclosure includes a combinationof one or more of the aforementioned modified nucleobases (e.g., acombination of 2, 3 or 4 of the aforementioned modified nucleobases).

In some embodiments, the modified nucleobase is pseudouridine (ψ),N1-methylpseudouridine (m¹ψ), 2-thiouridine, 4′-thiouridine,5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methoxyuridine, or 2′-O-methyl uridine. In some embodiments, an mmRNAof the disclosure includes a combination of one or more of theaforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 ofthe aforementioned modified nucleobases).

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine includeN4-acetyl-cytidine (ac⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C),1-methyl-pseudoisocytidine, 2-thio-cytidine (s²C),2-thio-5-methyl-cytidine. In some embodiments, an mmRNA, of thedisclosure includes a combination of one or more of the aforementionedmodified nucleobases (e.g., a combination of 2, 3 or 4 of theaforementioned modified nucleobases).

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include7-deaza-adenine, 1-methyl-adenosine (m¹A), 2-methyl-adenine (m²A),N6-methyl-adenosine (m⁶A). In some embodiments, an mmRNA, of thedisclosure includes a combination of one or more of the aforementionedmodified nucleobases (e.g., a combination of 2, 3 or 4 of theaforementioned modified nucleobases).

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine(mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ₀),7-aminomethyl-7-deaza-guanosine (preQ₁), 7-methyl-guanosine (m⁷G),1-methyl-guanosine (m¹G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine. Insome embodiments, an mmRNA of the disclosure includes a combination ofone or more of the aforementioned modified nucleobases (e.g., acombination of 2, 3 or 4 of the aforementioned modified nucleobases).

In some embodiments, the modified nucleobase is 1-methyl-pseudouridine(m¹ψ), 5-methoxy-uridine (mo⁵U), 5-methyl-cytidine (m⁵C), pseudouridine(ψ), α-thio-guanosine, or α-thio-adenosine. In some embodiments, anmmRNA of the disclosure includes a combination of one or more of theaforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 ofthe aforementioned modified nucleobases).

In some embodiments, the mmRNA, comprises pseudouridine (ψ). In someembodiments, the mmRNA, comprises pseudouridine (ψ) and5-methyl-cytidine (m⁵C). In some embodiments, the mmRNA, comprises1-methyl-pseudouridine (m¹ψ). In some embodiments, the mmRNA comprises1-methyl-pseudouridine (m¹ψ) and 5-methyl-cytidine (m⁵C). In someembodiments, the mmRNA, comprises 2-thiouridine (s²U). In someembodiments, the mmRNA, comprises 2-thiouridine and 5-methyl-cytidine(m⁵C). In some embodiments, the mmRNA, comprises 5-methoxy-uridine(mo⁵U). In some embodiments, the RNA, e.g., comprises 5-methoxy-uridine(mo⁵U) and 5-methyl-cytidine (m⁵C). In some embodiments, the mmRNA,comprises 2′-O-methyl uridine. In some embodiments, the mmRNA, comprises2′-O-methyl uridine and 5-methyl-cytidine (m⁵C). In some embodiments,the mmRNA, comprises N6-methyl-adenosine (m⁶A). In some embodiments, themmRNA, comprises N6-methyl-adenosine (m⁶A) and 5-methyl-cytidine (m⁵C).

In certain embodiments, an mmRNA, of the disclosure is uniformlymodified (i.e., fully modified, modified through-out the entiresequence) for a particular modification. For example, an mmRNA, can beuniformly modified with 1-methyl-pseudouridine (m¹ψ) or with5-methyl-cytidine (m⁵C), meaning that all uridine or cytosine residuesin the mmRNA, sequence are replaced with 1-methyl-pseudouridine (m¹ψ) orwith 5-methyl-cytidine (m⁵C), respectively. Similarly, mmRNAs, of thedisclosure can be uniformly modified for any type of nucleoside residuepresent in the sequence by replacement with a modified residue such asthose set forth above.

In some embodiments, an mRNA of the disclosure may be modified in acoding region (e.g., an open reading frame encoding a polypeptide). Inother embodiments, an mRNA may be modified in regions besides a codingregion. For example, in some embodiments, a 5′-UTR and/or a 3′-UTR areprovided, wherein either or both may independently contain one or moredifferent nucleoside modifications. In such embodiments, nucleosidemodifications may also be present in the coding region.

Examples of nucleoside modifications and combinations thereof that maybe present in mmRNAs, of the present disclosure include, but are notlimited to, those described in PCT Patent Application Publications:WO2012045075, WO2014081507, WO2014093924, WO2014164253, andWO2014159813.

The mmRNAs, of the disclosure can include a combination of modificationsto the sugar, the nucleobase, and/or the internucleoside linkage. Thesecombinations can include any one or more modifications described herein.

Examples of modified nucleosides and modified nucleoside combinationsare provided below in Table 1 and Table 2. These combinations ofmodified nucleotides can be used to form the mmRNAs, of the disclosure.In certain embodiments, the modified nucleosides may be partially orcompletely substituted for the natural nucleotides of the mmRNAs, of thedisclosure. As a non-limiting example, the natural nucleotide uridinemay be substituted with a modified nucleoside described herein. Inanother non-limiting example, the natural nucleoside uridine may bepartially substituted (e.g., about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or99.9% of the natural uridines) with at least one of the modifiednucleoside disclosed herein.

TABLE 1 Combinations of Nucleoside Modifications Modified NucleotideModified Nucleotide Combination α-thio-cytidineα-thio-cytidine/5-iodo-uridine α-thio-cytidine/N1-methyl-pseudouridineα-thio-cytidine/α-thio-uridine α-thio-cytidine/5-methyl-uridineα-thio-cytidine/pseudo-uridine about 50% of the cytosines areα-thio-cytidine pseudoisocytidine pseudoisocytidine/5-iodo-uridinepseudoisocytidine/N1-methyl-pseudouridinepseudoisocytidine/α-thio-uridine pseudoisocytidine/5-methyl-uridinepseudoisocytidine/pseudouridine about 25% of cytosines arepseudoisocytidine pseudoisocytidine/about 50% of uridines are N1-methyl-pseudouridine and about 50% of uridine are pseudouridinepseudoisocytidine/about 25% of uridines are N1- methyl-pseudouridine andabout 25% of uridines are pseudouridine pyrrolo-cytidinepyrrolo-cytidine/5-iodo-uridine pyrrolo-cytidine/N1-methyl-pseudouridinepyrrolo-cytidine/α-thio-uridine pyrrolo-cytidine/5-methyl-uridinepyrrolo-cytidine/pseudouridine about 50% of the cytosines arepyrrolo-cytidine 5-methyl-cytidine 5-methyl-cytidine/5-iodo-uridine5-methyl-cytidine/N1-methyl-pseudouridine5-methyl-cytidine/α-thio-uridine 5-methyl-cytidine/5-methyl-uridine5-methyl-cytidine/pseudouridine about 25% of cytosines are5-methyl-cytidine about 50% of cytosines are 5-methyl-cytidine5-methyl-cytidine/5-methoxy-uridine 5-methyl-cytidine/5-bromo-uridine5-methyl-cytidine/2-thio-uridine 5-methyl-cytidine/about 50% of uridinesare 2-thio-uridine about 50% of uridines are 5-methyl-cytidine/ about50% of uridines are 2-thio-uridine N4-acetyl-cytidineN4-acetyl-cytidine/5-iodo-uridineN4-acetyl-cytidine/N1-methyl-pseudouridineN4-acetyl-cytidine/α-thio-uridine N4-acetyl-cytidine/5-methyl-uridineN4-acetyl-cytidine/pseudouridine about 50% of cytosines areN4-acetyl-cytidine about 25% of cytosines are N4-acetyl-cytidineN4-acetyl-cytidine/5-methoxy-uridine N4-acetyl-cytidine/5-bromo-uridineN4-acetyl-cytidine/2-thio-uridine about 50% of cytosines areN4-acetyl-cytidine/ about 50% of uridines are 2-thio-uridine

TABLE 2 Modified Nucleosides and Combinations Thereof1-(2,2,2-Trifluoroethyl)pseudo-UTP 1-Ethyl-pseudo-UTP1-Methyl-pseudo-U-alpha-thio-TP 1-methyl-pseudouridine TP, ATP, GTP, CTP1-methyl-pseudo-UTP/5-methyl-CTP/ATP/GTP 1-Propyl-pseudo-UTP 25%5-Aminoallyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25%5-Aminoallyl-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Bromo-CTP +75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Bromo-CTP + 75% CTP/75%5-Methoxy-UTP + 25% UTP 25% 5-Bromo-CTP + 75% CTP/1-Methyl-pseudo-UTP25% 5-Carboxy-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25%5-Carboxy-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Ethyl-CTP +75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Ethyl-CTP + 75% CTP/75%5-Methoxy-UTP + 25% UTP 25% 5-Ethynyl-CTP + 75% CTP/25% 5-Methoxy-UTP +75% UTP 25% 5-Ethynyl-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25%5-Fluoro-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Fluoro-CTP +75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Formyl-CTP + 75% CTP/25%5-Methoxy-UTP + 75% UTP 25% 5-Formyl-CTP + 75% CTP/75% 5-Methoxy-UTP +25% UTP 25% 5-Hydroxymethyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP25% 5-Hydroxymethyl-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25%5-Iodo-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Iodo-CTP + 75%CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Methoxy-CTP + 75% CTP/25%5-Methoxy-UTP + 75% UTP 25% 5-Methoxy-CTP + 75% CTP/75% 5-Methoxy-UTP +25% UTP 25% 5-Methyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75%1-Methyl-pseudo-UTP 25% 5-Methyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75%UTP 25% 5-Methyl-CTP + 75% CTP/50% 5-Methoxy-UTP + 50%1-Methyl-pseudo-UTP 25% 5-Methyl-CTP + 75% CTP/50% 5-Methoxy-UTP + 50%UTP 25% 5-Methyl-CTP + 75% CTP/5-Methoxy-UTP 25% 5-Methyl-CTP + 75%CTP/75% 5-Methoxy-UTP + 25% 1-Methyl-pseudo-UTP 25% 5-Methyl-CTP + 75%CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Phenyl-CTP + 75% CTP/25%5-Methoxy-UTP + 75% UTP 25% 5-Phenyl-CTP + 75% CTP/75% 5-Methoxy-UTP +25% UTP 25% 5-Trifluoromethyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP25% 5-Trifluoromethyl-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25%5-Trifluoromethyl-CTP + 75% CTP/1-Methyl-pseudo-UTP 25% N4-Ac-CTP + 75%CTP/25% 5-Methoxy-UTP + 75% UTP 25% N4-Ac-CTP + 75% CTP/75%5-Methoxy-UTP + 25% UTP 25% N4-Bz-CTP + 75% CTP/25% 5-Methoxy-UTP + 75%UTP 25% N4-Bz-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25%N4-Methyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% N4-Methyl-CTP +75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% Pseudo-iso-CTP + 75% CTP/25%5-Methoxy-UTP + 75% UTP 25% Pseudo-iso-CTP + 75% CTP/75% 5-Methoxy-UTP +25% UTP 25% 5-Bromo-CTP/75% CTP/Pseudo-UTP 25% 5-methoxy-UTP/25%5-methyl-CTP/ATP/GTP 25% 5-methoxy-UTP/5-methyl-CTP/ATP/GTP 25%5-methoxy-UTP/75% 5-methyl-CTP/ATP/GTP 25% 5-methoxy-UTP/CTP/ATP/GTP 25%5-metoxy-UTP/50% 5-methyl-CTP/ATP/GTP 2-Amino-ATP 2-Thio-CTP2-thio-pseudouridine TP, ATP, GTP, CTP 2-Thio-pseudo-UTP 2-Thio-UTP3-Methyl-CTP 3-Methyl-pseudo-UTP 4-Thio-UTP 50% 5-Bromo-CTP + 50%CTP/1-Methyl-pseudo-UTP 50% 5-Hydroxymethyl-CTP + 50%CTP/1-Methyl-pseudo-UTP 50% 5-methoxy-UTP/5-methyl-CTP/ATP/GTP 50%5-Methyl-CTP + 50% CTP/25% 5-Methoxy-UTP + 75% 1-Methyl-pseudo-UTP 50%5-Methyl-CTP + 50% CTP/25% 5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP +50% CTP/50% 5-Methoxy-UTP + 50% 1-Methyl-pseudo-UTP 50% 5-Methyl-CTP +50% CTP/50% 5-Methoxy-UTP + 50% UTP 50% 5-Methyl-CTP + 50%CTP/5-Methoxy-UTP 50% 5-Methyl-CTP + 50% CTP/75% 5-Methoxy-UTP + 25%1-Methyl-pseudo-UTP 50% 5-Methyl-CTP + 50% CTP/75% 5-Methoxy-UTP + 25%UTP 50% 5-Trifluoromethyl-CTP + 50% CTP/1-Methyl-pseudo-UTP 50%5-Bromo-CTP/50% CTP/Pseudo-UTP 50% 5-methoxy-UTP/25%5-methyl-CTP/ATP/GTP 50% 5-methoxy-UTP/50% 5-methyl-CTP/ATP/GTP 50%5-methoxy-UTP/75% 5-methyl-CTP/ATP/GTP 50% 5-methoxy-UTP/CTP/ATP/GTP5-Aminoallyl-CTP 5-Aminoallyl-CTP/5-Methoxy-UTP 5-Aminoallyl-UTP5-Bromo-CTP 5-Bromo-CTP/5-Methoxy-UTP 5-Bromo-CTP/1-Methyl-pseudo-UTP5-Bromo-CTP/Pseudo-UTP 5-bromocytidine TP, ATP, GTP, UTP 5-Bromo-UTP5-Carboxy-CTP/5-Methoxy-UTP 5-Ethyl-CTP/5-Methoxy-UTP5-Ethynyl-CTP/5-Methoxy-UTP 5-Fluoro-CTP/5-Methoxy-UTP5-Formyl-CTP/5-Methoxy-UTP 5-Hydroxy-methyl-CTP/5-Methoxy-UTP5-Hydroxymethyl-CTP 5-Hydroxymethyl-CTP/1-Methyl-pseudo-UTP5-Hydroxymethyl-CTP/5-Methoxy-UTP 5-hydroxymethyl-cytidine TP, ATP, GTP,UTP 5-Iodo-CTP/5-Methoxy-UTP 5-Me-CTP/5-Methoxy-UTP 5-Methoxy carbonylmethyl-UTP 5-Methoxy-CTP/5-Methoxy-UTP 5-methoxy-uridine TP, ATP, GTP,UTP 5-methoxy-UTP 5-Methoxy-UTP 5-Methoxy-UTP/N6-Isopentenyl-ATP5-methoxy-UTP/25% 5-methyl-CTP/ATP/GTP5-methoxy-UTP/5-methyl-CTP/ATP/GTP 5-methoxy-UTP/75%5-methyl-CTP/ATP/GTP 5-methoxy-UTP/CTP/ATP/GTP 5-Methyl-2-thio-UTP5-Methylaminomethyl-UTP 5-Methyl-CTP/5-Methoxy-UTP5-Methyl-CTP/5-Methoxy-UTP(cap 0) 5-Methyl-CTP/5-Methoxy-UTP(No cap)5-Methyl-CTP/25% 5-Methoxy-UTP + 75% 1-Methyl-pseudo-UTP5-Methyl-CTP/25% 5-Methoxy-UTP + 75% UTP 5-Methyl-CTP/50%5-Methoxy-UTP + 50% 1-Methyl-pseudo-UTP 5-Methyl-CTP/50% 5-Methoxy-UTP +50% UTP 5-Methyl-CTP/5-Methoxy-UTP/N6-Me-ATP 5-Methyl-CTP/75%5-Methoxy-UTP + 25% 1-Methyl-pseudo-UTP 5-Methyl-CTP/75% 5-Methoxy-UTP +25% UTP 5-Phenyl-CTP/5-Methoxy-UTP 5-Trifluoro-methyl-CTP/5-Methoxy-UTP5-Trifluoromethyl-CTP 5-Trifluoromethyl-CTP/5-Methoxy-UTP5-Trifluoromethyl-CTP/1-Methyl-pseudo-UTP5-Trifluoromethyl-CTP/Pseudo-UTP 5-Trifluoromethyl-UTP5-trifluromethylcytidine TP, ATP, GTP, UTP 75% 5-Aminoallyl-CTP + 25%CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Aminoallyl-CTP + 25% CTP/75%5-Methoxy-UTP + 25% UTP 75% 5-Bromo-CTP + 25% CTP/25% 5-Methoxy-UTP +75% UTP 75% 5-Bromo-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75%5-Carboxy-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Carboxy-CTP +25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Ethyl-CTP + 25% CTP/25%5-Methoxy-UTP + 75% UTP 75% 5-Ethyl-CTP + 25% CTP/75% 5-Methoxy-UTP +25% UTP 75% 5-Ethynyl-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75%5-Ethynyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Fluoro-CTP +25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Fluoro-CTP + 25% CTP/75%5-Methoxy-UTP + 25% UTP 75% 5-Formyl-CTP + 25% CTP/25% 5-Methoxy-UTP +75% UTP 75% 5-Formyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75%5-Hydroxymethyl-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75%5-Hydroxymethyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75%5-Iodo-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Iodo-CTP + 25%CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Methoxy-CTP + 25% CTP/25%5-Methoxy-UTP + 75% UTP 75% 5-Methoxy-CTP + 25% CTP/75% 5-Methoxy-UTP +25% UTP 75% 5-methoxy-UTP/5-methyl-CTP/ATP/GTP 75% 5-Methyl-CTP + 25%CTP/25% 5-Methoxy-UTP + 75% 1-Methyl-pseudo-UTP 75% 5-Methyl-CTP + 25%CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP/50%5-Methoxy-UTP + 50% 1-Methyl-pseudo-UTP 75% 5-Methyl-CTP + 25% CTP/50%5-Methoxy-UTP + 50% UTP 75% 5-Methyl-CTP + 25% CTP/5-Methoxy-UTP 75%5-Methyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% 1-Methyl-pseudo-UTP 75%5-Methyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Phenyl-CTP +25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Phenyl-CTP + 25% CTP/75%5-Methoxy-UTP + 25% UTP 75% 5-Trifluoromethyl-CTP + 25% CTP/25%5-Methoxy-UTP + 75% UTP 75% 5-Trifluoromethyl-CTP + 25% CTP/75%5-Methoxy-UTP + 25% UTP 75% 5-Trifluoromethyl-CTP + 25%CTP/1-Methyl-pseudo-UTP 75% N4-Ac-CTP + 25% CTP/25% 5-Methoxy-UTP + 75%UTP 75% N4-Ac-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% N4-Bz-CTP +25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% N4-Bz-CTP + 25% CTP/75%5-Methoxy-UTP + 25% UTP 75% N4-Methyl-CTP + 25% CTP/25% 5-Methoxy-UTP +75% UTP 75% N4-Methyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75%Pseudo-iso-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75%Pseudo-iso-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Bromo-CTP/25%CTP/1-Methyl-pseudo-UTP 75% 5-Bromo-CTP/25% CTP/Pseudo-UTP 75%5-methoxy-UTP/25% 5-methyl-CTP/ATP/GTP 75% 5-methoxy-UTP/50%5-methyl-CTP/ATP/GTP 75% 5-methoxy-UTP/75% 5-methyl-CTP/ATP/GTP 75%5-methoxy-UTP/CTP/ATP/GTP 8-Aza-ATP Alpha-thio-CTP CTP/25%5-Methoxy-UTP + 75% 1-Methyl-pseudo-UTP CTP/25% 5-Methoxy-UTP + 75% UTPCTP/50% 5-Methoxy-UTP + 50% 1-Methyl-pseudo-UTP CTP/50% 5-Methoxy-UTP +50% UTP CTP/5-Methoxy-UTP CTP/5-Methoxy-UTP CTP/5-Methoxy-UTP (cap 0)CTP/5-Methoxy-UTP(No cap) CTP/75% 5-Methoxy-UTP + 25%1-Methyl-pseudo-UTP CTP/75% 5-Methoxy-UTP + 25% UTP CTP/UTP(No cap)N1-Me-GTP N4-Ac-CTP N4Ac-CTP/1-Methyl-pseudo-UTP N4Ac-CTP/5-Methoxy-UTPN4-acetyl-cytidine TP, ATP, GTP, UTP N4-Bz-CTP/5-Methoxy-UTP N4-methylCTP N4-Methyl-CTP/5-Methoxy-UTP Pseudo-iso-CTP/5-Methoxy-UTPPseudoU-alpha-thio-TP pseudouridine TP, ATP, GTP, CTPpseudo-UTP/5-methyl-CTP/ATP/GTP UTP-5-oxyacetic acid Me ester Xanthosine

According to the disclosure, polynucleotides of the disclosure may besynthesized to comprise the combinations or single modifications ofTable 1 or Table 2.

Where a single modification is listed, the listed nucleoside ornucleotide represents 100 percent of that A, U, G or C nucleotide ornucleoside having been modified. Where percentages are listed, theserepresent the percentage of that particular A, U, G or C nucleobasetriphosphate of the total amount of A, U, G, or C triphosphate present.For example, the combination: 25% 5-Aminoallyl-CTP+75% CTP/25%5-Methoxy-UTP+75% UTP refers to a polynucleotide where 25% of thecytosine triphosphates are 5-Aminoallyl-CTP while 75% of the cytosinesare CTP; whereas 25% of the uracils are 5-methoxy UTP while 75% of theuracils are UTP. Where no modified UTP is listed then the naturallyoccurring ATP, UTP, GTP and/or CTP is used at 100% of the sites of thosenucleotides found in the polynucleotide. In this example all of the GTPand ATP nucleotides are left unmodified.

The mRNAs of the present disclosure, or regions thereof, may be codonoptimized. Codon optimization methods are known in the art and may beuseful for a variety of purposes: matching codon frequencies in hostorganisms to ensure proper folding, bias GC content to increase mRNAstability or reduce secondary structures, minimize tandem repeat codonsor base runs that may impair gene construction or expression, customizetranscriptional and translational control regions, insert or removeproteins trafficking sequences, remove/add post translation modificationsites in encoded proteins (e.g., glycosylation sites), add, remove orshuffle protein domains, insert or delete restriction sites, modifyribosome binding sites and mRNA degradation sites, adjust translationrates to allow the various domains of the protein to fold properly, orto reduce or eliminate problem secondary structures within thepolynucleotide. Codon optimization tools, algorithms and services areknown in the art; non-limiting examples include services from GeneArt(Life Technologies), DNA2.0 (Menlo Park, Calif.) and/or proprietarymethods. In one embodiment, the mRNA sequence is optimized usingoptimization algorithms, e.g., to optimize expression in mammalian cellsor enhance mRNA stability.

In certain embodiments, the present disclosure includes polynucleotideshaving at least 80%, at least 85%, at least 90%, at least 95%, at least98%, or at least 99% sequence identity to any of the polynucleotidesequences described herein.

The mRNAs, of the present disclosure may be produced by means availablein the art, including but not limited to in vitro transcription (IVT)and synthetic methods. Enzymatic (IVT), solid-phase, liquid-phase,combined synthetic methods, small region synthesis, and ligation methodsmay be utilized. In one embodiment, mRNAs, are made using IVT enzymaticsynthesis methods. Methods of making polynucleotides by IVT are known inthe art and are described in International Application PCT/US2013/30062,the contents of which are incorporated herein by reference in theirentirety. Accordingly, the present disclosure also includespolynucleotides, e.g., DNA, constructs and vectors that may be used toin vitro transcribe an mRNA described herein.

Non-natural modified nucleobases may be introduced into polynucleotides,e.g., mRNA, during synthesis or post-synthesis. In certain embodiments,modifications may be on internucleoside linkages, purine or pyrimidinebases, or sugar. In particular embodiments, the modification may beintroduced at the terminal of a polynucleotide chain or anywhere else inthe polynucleotide chain; with chemical synthesis or with a polymeraseenzyme. Examples of modified nucleic acids and their synthesis aredisclosed in PCT application No. PCT/US2012/058519. Synthesis ofmodified polynucleotides is also described in Verma and Eckstein, AnnualReview of Biochemistry, vol. 76, 99-134 (1998).

Either enzymatic or chemical ligation methods may be used to conjugatepolynucleotides or their regions with different functional moieties,such as targeting or delivery agents, fluorescent labels, liquids,nanoparticles, etc. Conjugates of polynucleotides and modifiedpolynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol.1(3), 165-187 (1990).

microRNA Binding Sites for miRs Expressed in Immune Cells

microRNAs (or miRNA) are 19-25 nucleotide long (commonly 19-23nucleotides long, most typically 22 nucleotides long) noncoding RNAsthat bind to the 3′UTR of nucleic acid molecules andpost-translationally down-regulate gene expression either by reducingnucleic acid molecule stability or by inhibiting translation. The mRNAsof the disclosure may comprise one or more microRNA target sequences orsites, microRNA binding sequences or sites, sequence complementary to amicroRNA sequences, or sequence complementary to a microRNA seed regionor sequence. Such sequences may correspond to any known microRNA such asthose taught in US Publication US2005/0261218 and US PublicationUS2005/0059005, the contents of which are incorporated herein byreference in their entirety. A microRNA sequence comprises a “seed”region or sequence, i.e., a sequence in the region of positions 2-8 ofthe mature microRNA, which sequence has perfect Watson-Crickcomplementarity to the miRNA target sequence. The bases of the microRNAseed region or sequence have complete complementarity with the targetsequence. microRNAs derive enzymatically from regions of RNA transcriptsthat fold back on themselves to form short hairpin structures oftentermed a pre-miRNA (precursor-miRNA). The pre-miRNA typically has atwo-nucleotide overhang at its 3′ end, and has 3′ hydroxyl and 5′phosphate groups. This precursor-mRNA is processed in the nucleus andsubsequently transported to the cytoplasm where it is further processedby DICER (a RNase III enzyme), to form a mature microRNA ofapproximately 22 nucleotides. The mature microRNA is then incorporatedinto a ribonuclear particle to form the RNA-induced silencing complex,RISC, which mediates gene silencing. Art-recognized nomenclature formature miRNAs typically designates the arm of the pre-miRNA from whichthe mature miRNA derives; “5p” means the microRNA is from the 5 primearm of the pre-miRNA hairpin and “3p” means the microRNA is from the 3prime end of the pre-miRNA hairpin. A miR referred to by number hereincan refer to either of the two mature microRNAs originating fromopposite arms of the same pre-miRNA (e.g., either the 3p or 5pmicroRNA). All miRs referred to herein are intended to include both the3p and 5p arms/sequences, unless particularly specified by the 3p or 5pdesignation.

In some embodiments, an mRNA of the disclosure may include one or moremicroRNA (miRNA) binding sites. As used herein, the term “microRNA(miRNA) binding site” refers to a sequence within a polynucleotide,e.g., within a DNA or within an RNA transcript, that has sufficientcomplementarity to all or a region of a miRNA to interact with,associate with or bind to the miRNA. In exemplary embodiments, miRNAbinding sites are included in mRNAs, for example, in the 5′ UTR and/or3′ UTR of an mRNA. A miR binding site sequence having sufficientcomplementarity to the miR refers to a degree of complementaritysufficient to facilitate miR-mediated regulation of the mRNA, e.g.,miR-mediated translational repression or degradation of the mRNA. Inexemplary aspects of the disclosure, a miR binding site sequence havingsufficient complementarity to the miR refers to a degree ofcomplementarity sufficient to facilitate miR-mediated degradation of themRNA, e.g., miR-guided RISC-mediated cleavage of mRNA. The miR bindingsite can have complementarity to, for example, a 19-25 nucleotide longmiR sequence, to a 19-23 nucleotide long miR, most typically to a 22nucleotide long miR sequence. A miR binding site may be complementary toonly a portion of a miR, e.g., to a portion 1, 2, 3 or 4 nucleotidesshorter that a naturally-occurring miR. Full or complete complementarity(e.g., fully complementary or completely complementary over all or asignificant portion of a naturally-occurring miR) is preferred when thedesired regulation is mRNA degradation. In some embodiments, a miRNAbinding site includes a sequence that has complementarity (e.g., partialor complete complementarity) with an miRNA seed sequence. In particularembodiments, the miRNA binding site includes a sequence that hascomplete complementarity with a miRNA seed sequence. In someembodiments, a miRNA binding site includes a sequence that hascomplementarity (e.g., partial or complete complementarity) with a miRNAsequence. In particular embodiments, the miRNA binding site includes asequence that has complete complementarity with a miRNA sequence. Insome embodiments, a miRNA binding site has complete complementarity witha miRNA sequence but for 1, 2 or 3 nucleotide substitutions, terminaladditions, and/or truncations.

One or more miR binding sequences can be incorporated in an mRNA of thedisclosure for one or more of a variety of different purposes. Forexample, incorporation of one or more miRNA binding sites into an mRNAof the disclosure may target the molecule for degradation or reducedtranslation, provided the miRNA in question is available (e.g.,expressed in a target cell or tissue.) In some embodiments,incorporation of one or more miRNA binding sites into an mRNA of thedisclosure may reduce the hazard of off-target effects upon nucleic acidmolecule delivery and/or enable tissue-specific regulation of expressionof a polypeptide encoded by the mRNA. In yet other embodiments,incorporation of one or more miRNA binding sites into an mRNA of thedisclosure can modulate immune responses upon nucleic acid delivery invivo. In further embodiments, incorporation of one or more miRNA bindingsites into an mRNA of the disclosure can modulate accelerated bloodclearance (ABC) of lipid-comprising compounds and compositions describedherein.

Representative miRNAs were selected based on expression and abundance inimmune cells of the hematopoietic lineage, such as B cells, T cells,macrophages, dendritic cells, and cells that are known to expressTLR7/TLR8 and/or able to secrete cytokines such as endothelial cells andplatelets. The miRNA set thus included miRs that may be responsible inpart for the immunogenicity of these cells, and such that acorresponding miR-site incorporation in the mRNA could lead todestabilization of the mRNA and/or suppression of translation from thesemRNAs in the specific cell type. Non-limiting representative examplesinclude miR-142, miR-144, miR-150, miR-155 and miR-223, which arespecific for many of the hematopoietic cells; miR-142, miR150, miR-16and miR-223, which are expressed in B cells; miR-223, miR-451, miR-26a,miR-16, which are expressed in progenitor hematopoietic cells; andmiR-126, which is expressed in plasmacytoid dendritic cells, plateletsand endothelial cells. For further discussion of tissue expression ofmiRs see e.g., Teruel-Montoya, R. et al. (2014) PLoS One 9:e102259;Landgraf, P. et al. (2007) Cell 129:1401-1414; Bissels, U. et al. (2009)RNA 15:2375-2384. As is evidenced, any one miR-site incorporation in the3′UTR and/or 5′ UTR may mediate such effects in multiple cell types ofinterest (e.g., miR-142 is abundant in both B cells and dendriticcells).

It is beneficial to target the same cell type with multiple miRs and toincorporate binding sites to each of the 3p and 5p arm if both areabundant (e.g., both miR-142-3p and miR142-5p are abundant inhematopoietic stem cells). Thus, for example, in certain embodiments, anmRNA construct contains two or more (e.g., two, three, four or more) miRbindings sites from: (i) the group consisting of miR-142, miR-144,miR-150, miR-155 and miR-223 (which are expressed in many hematopoieticcells); or (ii) the group consisting of miR-142, miR150, miR-16 andmiR-223 (which are expressed in B cells); or the group consisting ofmiR-223, miR-451, miR-26a, miR-16 (which are expressed in progenitorhematopoietic cells).

It is also beneficial to combine various miRs such that multiple celltypes of interest are targeted at the same time (e.g., miR-142 andmiR-126 to target many cells of the hematopoietic lineage andendothelial cells). Thus, for example, in certain embodiments, an mRNAconstruct contains two or more (e.g., two, three, four or more) miRbindings sites, wherein: (i) at least one of the miRs targets cells ofthe hematopoietic lineage (e.g., miR-142, miR-144, miR-150, miR-155 ormiR-223) and at least one of the miRs targets plasmacytoid dendriticcells, platelets or endothelial cells (e.g., miR-126); or (ii) at leastone of the miRs targets B cells (e.g., miR-142, miR150, miR-16 ormiR-223) and at least one of the miRs targets plasmacytoid dendriticcells, platelets or endothelial cells (e.g., miR-126); or (iii) at leastone of the miRs targets progenitor hematopoietic cells (e.g., miR-223,miR-451, miR-26a or miR-16) and at least one of the miRs targetsplasmacytoid dendritic cells, platelets or endothelial cells (e.g.,miR-126); or (iv) at least one of the miRs targets cells of thehematopoietic lineage (e.g., miR-142, miR-144, miR-150, miR-155 ormiR-223), at least one of the miRs targets B cells (e.g., miR-142,miR150, miR-16 or miR-223) and at least one of the miRs targetsplasmacytoid dendritic cells, platelets or endothelial cells (e.g.,miR-126); or any other possible combination of the foregoing fourclasses of miR binding sites (i.e., those targeting the hematopoieticlineage, those targeting B cells, those targeting progenitorhematopoietic cells and/or those targeting plamacytoid dendriticcells/platelets/endothelial cells).

Accordingly, in one embodiment, to modulate immune responses, an mRNAcan comprise one or more miR binding sequences that bind to one or moremiRs that are expressed in conventional immune cells or any cell thatexpresses TLR7 and/or TLR8 and secrete pro-inflammatory cytokines and/orchemokines (e.g., in immune cells of peripheral lymphoid organs and/orsplenocytes and/or endothelial cells). It has now been discovered thatincorporation into an mRNA of one or more miRs that are expressed inconventional immune cells or any cell that expresses TLR7 and/or TLR8and secrete pro-inflammatory cytokines and/or chemokines (e.g., inimmune cells of peripheral lymphoid organs and/or splenocytes and/orendothelial cells) reduces or inhibits immune cell activation (e.g., Bcell activation, as measured by frequency of activated B cells) and/orcytokine production (e.g., production of IL-6, IFN-γ and/or TNFα).Furthermore, it has now been discovered that incorporation into an mRNAof one or more miRs that are expressed in conventional immune cells orany cell that expresses TLR7 and/or TLR8 and secrete pro-inflammatorycytokines and/or chemokines (e.g., in immune cells of peripherallymphoid organs and/or splenocytes and/or endothelial cells) can reduceor inhibit an anti-drug antibody (ADA) response against a protein ofinterest encoded by the mRNA.

In another embodiment, to modulate accelerated blood clearance of anmRNA delivered in a lipid-comprising compound or composition, the mRNAcan comprise one or more miR binding sequences that bind to one or moremiRs expressed in conventional immune cells or any cell that expressesTLR7 and/or TLR8 and secrete pro-inflammatory cytokines and/orchemokines (e.g., in immune cells of peripheral lymphoid organs and/orsplenocytes and/or endothelial cells). It has now been discovered thatincorporation into an mRNA of one or more miR binding sites reduces orinhibits accelerated blood clearance (ABC) of the lipid-comprisingcompound or composition for use in delivering the mRNA. Furthermore, ithas now been discovered that incorporation of one or more miR bindingsites into an mRNA reduces serum levels of anti-PEG anti-IgM (e.g,reduces or inhibits the acute production of IgMs that recognizepolyethylene glycol (PEG) by B cells) and/or reduces or inhibitsproliferation and/or activation of plasmacytoid dendritic cellsfollowing administration of a lipid-comprising compound or compositioncomprising the mRNA.

Such miR sequences may correspond to any known microRNA expressed inimmune cells, including but not limited to those taught in USPublication US2005/0261218 and US Publication US2005/0059005, thecontents of which are incorporated herein by reference in theirentirety. Non-limiting examples of miRs expressed in immune cellsinclude those expressed in spleen cells, myeloid cells, dendritic cells,plasmacytoid dendritic cells, B cells, T cells and/or macrophages. Forexample, miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24 andmiR-27 are expressed in myeloid cells, miR-155 is expressed in dendriticcells, B cells and T cells, miR-146 is upregulated in macrophages uponTLR stimulation and miR-126 is expressed in plasmacytoid dendriticcells. In certain embodiments, the miR(s) is expressed abundantly orpreferentially in immune cells. For example, miR-142 (miR-142-3p and/ormiR-142-5p), miR-126 (miR-126-3p and/or miR-126-5p), miR-146 (miR-146-3pand/or miR-146-5p) and miR-155 (miR-155-3p and/or miR155-5p) areexpressed abundantly in immune cells. These microRNA sequences are knownin the art and, thus, one of ordinary skill in the art can readilydesign binding sequences or target sequences to which these microRNAswill bind based upon Watson-Crick complementarity.

Accordingly, in various embodiments, the mRNA comprises at least onemicroRNA binding site for a miR selected from the group consisting ofmiR-142, miR-146, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24 andmiR-27. In another embodiment, the mRNA comprises at least two miRbinding sites for microRNAs expressed in immune cells. In variousembodiments, the mRNA comprises 1-4, one, two, three or four miR bindingsites for microRNAs expressed in immune cells. In another embodiment,the mRNA comprises three miR binding sites. These miR binding sites canbe for microRNAs selected from the group consisting of miR-142, miR-146,miR-155, miR-126, miR-16, miR-21, miR-223, miR-24, miR-27, andcombinations thereof. In one embodiment, the mRNA comprises two or more(e.g., two, three, four) copies of the same miR binding site expressedin immune cells, e.g., two or more copies of a miR binding site selectedfrom the group of miRs consisting of miR-142, miR-146, miR-155, miR-126,miR-16, miR-21, miR-223, miR-24, miR-27.

For example, in one embodiment, the mRNA comprises three copies of thesame miR binding site. As described in Example 8, in certainembodiments, use of three copies of the same miR binding site canexhibit beneficial properties as compared to use of a single miR bindingsite. Non-limiting examples of sequences for 3′ UTRs containing threemiR bindings sites are shown in SEQ ID NO: 38 (three miR-142-3p bindingsites), SEQ ID NO: 40 (three miR-142-5p binding sites) and SEQ ID NO: 54(three miR-122 binding sites).

In another embodiment, the mRNA comprises two or more (e.g., two, three,four) copies of at least two different miR binding sites expressed inimmune cells. Non-limiting examples of sequences of 3′ UTRs containingtwo or more different miR binding sites are shown in SEQ ID NO: 33 (onemiR-142-3p binding site and one miR-126-3p binding site), SEQ ID NO: 47(one miR-142-3p binding site and one miR-122-5p binding site), SEQ IDNO: 41 (two miR-142-5p binding sites and one miR-142-3p binding sites)and SEQ ID NO: 44 (two miR-155-5p binding sites and one miR-142-3pbinding sites).

In another embodiment, the mRNA comprises at least two miR binding sitesfor microRNAs expressed in immune cells, wherein one of the miR bindingsites is for miR-142-3p. In various embodiments, the mRNA comprisesbinding sites for miR-142-3p and miR-155 (miR-155-3p or miR-155-5p),miR-142-3p and miR-146 (miR-146-3 or miR-146-5p), or miR-142-3p andmiR-126 (miR-126-3p or miR-126-5p).

In another embodiment, the mRNA comprises at least two miR binding sitesfor microRNAs expressed in immune cells, wherein one of the miR bindingsites is for miR-126-3p. In various embodiments, the mRNA comprisesbinding sites for miR-126-3p and miR-155 (miR-155-3p or miR-155-5p),miR-126-3p and miR-146 (miR-146-3p or miR-146-5p), or miR-126-3p andmiR-142 (miR-142-3p or miR-142-5p).

In another embodiment, the mRNA comprises at least two miR binding sitesfor microRNAs expressed in immune cells, wherein one of the miR bindingsites is for miR-142-5p. In various embodiments, the mRNA comprisesbinding sites for miR-142-5p and miR-155 (miR-155-3p or miR-155-5p),miR-142-5p and miR-146 (miR-146-3 or miR-146-5p), or miR-142-5p andmiR-126 (miR-126-3p or miR-126-5p).

In yet another embodiment, the mRNA comprises at least two miR bindingsites for microRNAs expressed in immune cells, wherein one of the miRbinding sites is for miR-155-5p. In various embodiments, the mRNAcomprises binding sites for miR-155-5p and miR-142 (miR-142-3p ormiR-142-5p), miR-155-5p and miR-146 (miR-146-3 or miR-146-5p), ormiR-155-5p and miR-126 (miR-126-3p or miR-126-5p).

In exemplary embodiments, the one or more miR binding sites arepositioned within the 3′UTR, the 5′ UTR, or both the 3′ and 5′ UTRs,such that the mRNA has the desired properties. The miR binding site canbe positioned within the 3′ UTR immediately following the stop codon ofthe coding region within the mRNA construct (or, if there are multiplecopies of a stop codon in the construct, immediately following the finalstop codon) or the miR binding site(s) can be positioned furtherdownstream of the stop codon, in which case there are 3′ UTR basesbetween the stop codon and the miR binding site(s). For example, threenon-limiting examples of possible insertion sites for a miR in a 3′ UTRare shown in SEQ ID NOs: 48, 49 and 50, which show a 3′ UTR sequencewith a miR-142-3p site inserted in one of three different possibleinsertion sites, respectively, within the 3′ UTR. Furthermore, one ormore miR binding sites can be positioned within the 5′ UTR at one ormore possible insertion sites. For example, three non-limiting examplesof possible insertion sites for a miR in a 5′ UTR are described furtherin Example 9 and shown in SEQ ID NOs: 55, 56 and 57, which show a 5′ UTRsequence with a miR-142-3p site inserted into one of three differentpossible insertion sites, respectively, within the 5′ UTR. Additionally,SEQ ID NOs: 58, 59 and 60 show a 5′ UTR sequence with a miR-122 siteinserted into one of three different possible insertion sites,respectively, within the 5′ UTR.

In one embodiment, a codon optimized open reading frame encoding apolypeptide of interest comprises a stop codon and the at least onemicroRNA binding site is located within the 3′ UTR 1-100 nucleotidesafter the stop codon. In one embodiment, the codon optimized openreading frame encoding the polypeptide of interest comprises a stopcodon and the at least one microRNA binding site for a miR expressed inimmune cells is located within the 3′ UTR 30-50 nucleotides after thestop codon. In another embodiment, the codon optimized open readingframe encoding the polypeptide of interest comprises a stop codon andthe at least one microRNA binding site for a miR expressed in immunecells is located within the 3′ UTR at least 50 nucleotides after thestop codon. In other embodiments, the codon optimized open reading frameencoding the polypeptide of interest comprises a stop codon and the atleast one microRNA binding site for a miR expressed in immune cells islocated within the 3′ UTR immediately after the stop codon, or withinthe 3′ UTR 15-20 nucleotides after the stop codon or within the 3′ UTR70-80 nucleotides after the stop codon. In other embodiments, the 3′UTRcomprises more than one miR binding site (e.g., 2-4 miR binding sites),wherein there can be a spacer region (e.g., of 10-100, 20-70 or 30-50nucleotides in length) between each miR binding site. In anotherembodiment, the 3′ UTR comprises a spacer region between the end of themiR binding site(s) and the poly A tail nucleotides. For example, aspacer region of 10-100, 20-70 or 30-50 nucleotides in length can besituated between the end of the miR binding site(s) and the beginning ofthe poly A tail.

In one embodiment, a codon optimized open reading frame encoding apolypeptide of interest comprises a start codon and the at least onemicroRNA binding site is located within the 5′ UTR 1-100 nucleotidesbefore (upstream of) the start codon. In one embodiment, the codonoptimized open reading frame encoding the polypeptide of interestcomprises a start codon and the at least one microRNA binding site for amiR expressed in immune cells is located within the 5′ UTR 10-50nucleotides before (upstream of) the start codon. In another embodiment,the codon optimized open reading frame encoding the polypeptide ofinterest comprises a start codon and the at least one microRNA bindingsite for a miR expressed in immune cells is located within the 5′ UTR atleast 25 nucleotides before (upstream of) the start codon. In otherembodiments, the codon optimized open reading frame encoding thepolypeptide of interest comprises a start codon and the at least onemicroRNA binding site for a miR expressed in immune cells is locatedwithin the 5′ UTR immediately before the start codon, or within the 5′UTR 15-20 nucleotides before the start codon or within the 5′ UTR 70-80nucleotides before the start codon. In other embodiments, the 5′UTRcomprises more than one miR binding site (e.g., 2-4 miR binding sites),wherein there can be a spacer region (e.g., of 10-100, 20-70 or 30-50nucleotides in length) between each miR binding site.

In one embodiment, the 3′ UTR comprises more than one stop codon,wherein at least one miR binding site is positioned downstream of thestop codons. For example, a 3′ UTR can comprise 1, 2 or 3 stop codons.Non-limiting examples of triple stop codons that can be used include:UGAUAAUAG, UGAUAGUAA, UAAUGAUAG, UGAUAAUAA, UGAUAGUAG, UAAUGAUGA,UAAUAGUAG, UGAUGAUGA, UAAUAAUAA and UAGUAGUAG. Within a 3′ UTR, forexample, 1, 2, 3 or 4 miR binding sites, e.g., miR-142-3p binding sites,can be positioned immediately adjacent to the stop codon(s) or at anynumber of nucleotides downstream of the final stop codon. When the 3′UTR comprises multiple miR binding sites, these binding sites can bepositioned directly next to each other in the construct (i.e., one afterthe other) or, alternatively, spacer nucleotides can be positionedbetween each binding site.

In one embodiment, the 3′ UTR comprises three stop codons with a singlemiR-142-3p binding site located downstream of the 3rd stop codon.Non-limiting examples of sequences of 3′ UTR having three stop codonsand a single miR-142-3p binding site located at different positionsdownstream of the final stop codon are shown in SEQ ID Nos: 31 and48-50.

In one embodiment, the mmRNA comprises a 5′ UTR, a codon optimized openreading frame encoding a polypeptide of interest, a 3′ UTR comprisingthe at least one microRNA binding site for a miR expressed in immunecells, and a 3′ tailing region of linked nucleosides. In variousembodiments, the 3′ UTR comprises 1-4, at least two, one, two, three orfour microRNA binding sites for miRs expressed in immune cells,preferably abundantly or preferentially expressed in immune cells.

In one embodiment, the at least one miR expressed in immune cells is amiR-142-3p microRNA binding site. In one embodiment, the miR-142-3pmicroRNA binding site comprises the sequence shown in SEQ ID NO: 3. Inone embodiment, the 3′ UTR of the mRNA comprising the miR-142-3pmicroRNA binding site comprises the sequence shown in SEQ ID NO: 2.

In one embodiment, the at least one miR expressed in immune cells is amiR-126 microRNA binding site. In one embodiment, the miR-126 bindingsite is a miR-126-3p binding site. In one embodiment, the miR-126-3pmicroRNA binding site comprises the sequence shown in SEQ ID NO: 26. Inone embodiment, the 3′ UTR of the mmRNA comprising the miR-126-3pmicroRNA binding site comprises the sequence shown in SEQ ID NO: 27.

Non-limiting exemplary sequences for miRs to which a microRNA bindingsite(s) of the disclosure can bind include the following: miR-142-3p(SEQ ID NO: 8), miR-142-5p (SEQ ID NO: 9), miR-146-3p (SEQ ID NO: 10),miR-146-5p (SEQ ID NO: 11), miR-155-3p (SEQ ID NO: 12), miR-155-5p (SEQID NO: 13), miR-126-3p (SEQ ID NO: 14), miR-126-5p (SEQ ID NO: 15),miR-16-3p (SEQ ID NO: 16), miR-16-5p (SEQ ID NO: 17), miR-21-3p (SEQ IDNO: 18), miR-21-5p (SEQ ID NO: 19), miR-223-3p (SEQ ID NO: 20),miR-223-5p (SEQ ID NO: 21), miR-24-3p (SEQ ID NO: 22), miR-24-5p (SEQ IDNO: 23), miR-27-3p (SEQ ID NO: 24) and miR-27-5p (SEQ ID NO: 25). Othersuitable miR sequences expressed in immune cells (e.g., abundantly orpreferentially expressed in immune cells) are known and available in theart, for example at the University of Manchester's microRNA database,miRBase. Sites that bind any of the aforementioned miRs can be designedbased on Watson-Crick complementarity to the miR, typically 100%complementarity to the miR, and inserted into an mRNA construct of thedisclosure as described herein.

In yet other embodiments, the therapeutic window and/or differentialexpression (e.g., tissue-specific expression) of a polypeptide of thedisclosure may be altered by incorporation of a miRNA binding site intoan mRNA encoding the polypeptide. Examples of tissues where microRNA areknown to regulate mRNA, and thereby protein expression, include, but arenot limited to, liver (e.g., miR-122), muscle (e.g., miR-133, miR-206,and miR-208), endothelial cells (e.g., miR-17-92, and miR-126), myeloidcells (e.g., miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24,and miR-27), adipose tissue (e.g., let-7, and miR-30c), heart (e.g.,miR-ld and miR-149), kidney (e.g., miR-192, miR-194, and miR-204), andlung epithelial cells (e.g., let-7, miR-133, and miR-126). Thus, invarious embodiments, an mRNA can comprise one or more binding site forany of the aforementioned miRs, alone or in combination, to regulatethereby regulate tissue expression of an encoded protein of interest.

For example, an mRNA may include one or more miRNA binding sites thatare bound by miRNAs that have higher expression in one tissue type ascompared to another. In another example, an mRNA may include one or moremiRNA binding sites that are bound by miRNAs that have lower expressionin a cancer cell as compared to a non-cancerous cell of the same tissueof origin. When present in a cancer cell that expresses low levels ofsuch an miRNA, the polypeptide encoded by the mRNA typically will showincreased expression. If the polypeptide is able to induce apoptosis,this may result in preferential cell killing of cancer cells as comparedto normal cells.

For example, liver cancer cells (e.g., hepatocellular carcinoma cells)typically express low levels of miR-122 as compared to normal livercells. Therefore, an mRNA encoding a polypeptide that includes at leastone miR-122 binding site (e.g., in the 3′-UTR of the mRNA) willtypically express comparatively low levels of the polypeptide in normalliver cells and comparatively high levels of the polypeptide in livercancer cells. If the polypeptide is able to induce apoptosis, this cancause preferential cell killing of liver cancer cells (e.g.,hepatocellular carcinoma cells) as compared to normal liver cells.

Accordingly, as a non-limiting example of incorporation a miR bindingsite(s) into a mRNA to modulate tissue expression of an encoded proteinof interest, mRNAs of the disclosure may include at least one miR-122binding site. For example, a mRNA of the disclosure may include amiR-122 binding site that includes a sequence with partial or completecomplementarity with a miR-122 seed sequence. In some embodiments, amiR-122 seed sequence may correspond to nucleotides 2-7 of a miR-122. Insome embodiments, a miR-122 seed sequence may be 5′-GGAGUG-3′. In someembodiments, a miR-122 seed sequence may be nucleotides 2-8 of amiR-122. In some embodiments, a miR-122 seed sequence may be5′-GGAGUGU-3′. In some embodiments, the miR-122 binding site includes anucleotide sequence of 5′-UAUUUAGUGUGAUAAUGGCGUU-3′ (SEQ ID NO: 45) or5′-CAAACACCAUUGUCACACUCCA-3′ (SEQ ID NO: 46) or a complement thereof. Insome embodiments, inclusion of at least one miR-122 binding site in anmRNA may dampen expression of a polypeptide encoded by the mRNA in anormal liver cell as compared to other cell types that express lowlevels of miR-122. In other embodiments, inclusion of at least onemiR-122 binding site in an mRNA may allow increased expression of apolypeptide encoded by the mRNA in a liver cancer cell (e.g., ahepatocellular carcinoma cell) as compared to a normal liver cell.

In yet another embodiment, the mRNA (e.g., the 3′ UTR thereof) cancomprise at least one miR binding site for a miR expressed in immunecells, to thereby reduce or inhibit immune activation (e.g., B cellactivation, cytokine production, ADA responses) upon nucleic aciddelivery in vivo, and can comprise at least one miR binding site formodulating tissue expression of an encoded protein of interest. Forexample, in one embodiment, the mRNA comprises a miR-122 binding site,to thereby allow increased expression of a polypeptide encoded by themRNA in a liver cancer cell (e.g., a hepatocellular carcinoma cell) ascompared to a normal liver cell, and also comprises one or more miRbinding sites for a miR expressed in immune cells, e.g., selected fromthe group consisting of miR-142, miR-146, miR-155, miR-126, miR-16,miR-21, miR-223, miR-24, miR-27.

In another embodiment, the mRNA (e.g., the 3′ UTR thereof) can compriseat least one miR binding site to thereby reduce or inhibit acceleratedblood clearance, for example by reducing or inhibiting production ofIgMs, e.g., against PEG, by B cells and/or reducing or inhibitingproliferation and/or activation of pDCs, and can comprise at least onemiR binding site for modulating tissue expression of an encoded proteinof interest. For example, in one embodiment, the mRNA comprises amiR-122 binding site, to thereby allow increased expression of apolypeptide encoded by the mRNA in a liver cancer cell (e.g., ahepatocellular carcinoma cell) as compared to a normal liver cell, andalso comprises one or more miR binding sites, e.g., selected from thegroup consisting of miR-142, miR-146, miR-155, miR-126, miR-16, miR-21,miR-223, miR-24, miR-27.

In one embodiment, the mRNA comprises a miR-122 binding site and amiR-142-3p binding site. In another embodiment, the mRNA comprises amiR-122 binding site and a miR-142-5p binding site. In anotherembodiment, the mRNA comprises a miR-122 binding site and a miR-126-3pbinding site. In another embodiment, the mRNA comprises a miR-122binding site and a miR-155-5p binding site. In another embodiment, themRNA comprises a miR-122 binding site and a miR-126-3p binding site. Inanother embodiment, the mRNA comprises a miR-122 binding site, a miR-142(miR-142-3p or 142-5p) binding site and a miR-126 (miR-126-3p ormiR-126-5p) binding site. In another embodiment, the mRNA comprises amiR-122 binding site, a miR-142 (miR-142-3p or 142-5p) binding site anda miR-155 (miR-155-3p or miR-155-5p) binding site. In anotherembodiment, the mRNA comprises a miR-122 binding site, a miR-126(miR-126-3p or 126-5p) binding site and a miR-155 (miR-155-3p ormiR-155-5p) binding site. In yet another embodiment, the mRNA comprisesa miR-122 binding site, a miR-142 (miR-142-3p or miR-142-5p) bindingsite, a miR-126 (miR-126-3p or 126-5p) binding site and a miR-155(miR-155-3p or miR-155-5p) binding site. In any of these embodiments,the miR-122 binding site can be a miR-122-5p binding site.

A non-limiting example of a 3′ UTR sequence that comprises both amiR-142-3p binding site and a miR-122-5p binding site is shown in SEQ IDNO: 47. The structure of the 3′ UTR of SEQ ID NO: 47 includes three stopcodons at it's 5′ end, followed immediately by a single miR-142-3pbinding site, followed downstream by spacer nucleotides and then asingle miR-122-5p binding site. The distance between the miR bindingsites (e.g., miR-142-3p and miR-122-5p) can vary considerably; a numberof different constructs have been tested with differing placement of thetwo miR binding sites and all have been functional. In certainembodiments, a nucleotide spacer is positioned between the two miRbinding sites of a sufficient length to allow binding of RISC to eachone. In one embodiment, the two miR binding sites are positioned about40 bases apart from each other and the overall length of the 3′ UTR isapproximately 100-110 bases.

Proteins of Interest

The mRNAs of the disclosure can encode a protein of interest, typicallya protein having therapeutic properties for use in a subject. Theprotein of interest can be essentially any protein that can be encodedby the mRNA. In particular, a protein of interest can be one thatstimulates immune cell activation (e.g., B cell activation), such aseliciting an anti-drug antibody (ADA) response in a subject and, thus,for which reducing or inhibiting immune cell activation (e.g., reducingthe ADA response) in the subject is desirable. In various embodiments,the protein of interest can be, for example, a therapeutic protein, acytokine, a growth factor, an antibody or a fusion protein. Non-limitingexamples of therapeutic proteins include, for example, blood factors(such as Factor VIII and Factor VII), complement factors, Low DensityLipoprotein Receptor (LDLR) and MUT1. Non-limiting examples of cytokinesinclude, for example, interleukins, interferons, chemokines, lymphokinesand the like. Non-limiting examples of growth factors includeerythropoietin, EGFs, PDGFs, FGFs, TGFs, IGFs, TNFs, CSFs, MCSFs, GMCSFsand the like. Non-limiting examples of antibodies include, for example,adalimumab, infliximab, rituximab, ipilimumab, tocilizumab, canakinumab,itolizumab, tralokinumab. Non-limiting examples of fusion proteinsinclude, for example, etanercept, abatacept and belatacept.

In one embodiment, the protein of interest is human erythropoietin. Inone embodiment, the mRNA encodes human erythropoietin and comprises amicroRNA binding site that binds miR-142-3p, such as the mRNA having thesequence shown in SEQ ID NO: 1. In another embodiment, the mRNA encodeshuman erythropoietin and comprises a microRNA binding site that bindsmiR-126, such as the mRNA having the sequence shown in SEQ ID NO: 28. Inyet another embodiment, the mRNA encodes human erythropoietin andcomprises a microRNA binding site that binds miR-142-3p and a microRNAbinding site that binds miR-126, such as the mRNA having the sequenceshown in SEQ ID NO: 29. In another embodiment, the protein of interestis LDLR (for use in inhibiting cholesterol). In another embodiment, theprotein of interest is MUT1 (for use in the treatment of methylmalonicacidemia (MMA)). In yet other embodiments, the protein of interestencoded by the mmRNA is a therapeutic antibody, including but notlimited to the antibodies listed above.

Nanoparticles

The mRNAs, of the disclosure may be formulated in nanoparticles or otherdelivery vehicles, e.g., to protect them from degradation when deliveredto a subject. Illustrative nanoparticles are described in Panyam, J. &Labhasetwar, V. (2003) Adv. Drug Deliv. Rev. 55, 329-347 and Peer, D. etal. (2007) Nature Nanotech. 2, 751-760. In certain embodiments, an RNA,e.g., mRNA, of the disclosure is encapsulated within a nanoparticle. Inparticular embodiments, a nanoparticle is a particle having at least onedimension (e.g., a diameter) less than or equal to 1000 nM, less than orequal to 500 nM or less than or equal to 100 nM. In particularembodiments, a nanoparticle includes a lipid. Lipid nanoparticlesinclude, but are not limited to, liposomes and micelles. Any of a numberof lipids may be present, including cationic and/or ionizable lipids,anionic lipids, neutral lipids, amphipathic lipids, PEGylated lipids,and/or structural lipids. Such lipids can be used alone or incombination. In particular embodiments, a lipid nanoparticle comprisesone or more RNAs, e.g., mRNAs, described herein, e.g., a mmRNA encodinga polypeptide of interest and comprising at least microRNA one bindingsite for a miR expressed in immune cells.

In some embodiments, the lipid nanoparticle formulations of the mRNAs,described herein may include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or8) cationic and/or ionizable lipids. Such cationic lipids include, butare not limited to,3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10),N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine(KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25),1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate(DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane(DLin-KC2-DMA),2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA), (2R)-2-({8-[(3(3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2R)), (25)-2-({8-[(3(3)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(Octyl-CLinDMA (2S)).N,N-dioleyl-N,N-dimethylammonium chloride(“DODAC”); N-(2,3-dioleyloxy)propyl-N,N—N-triethylammonium chloride(“DOTMA”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”);1,2-Dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP.Cl”);3-β-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”),N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-ammoniumtrifluoracetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine(“DOGS”), 1,2-dioleoyl-3-dimethylammonium propane (“DODAP”),N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”). Additionally, a number of commercial preparations ofcationic and/or ionizable lipids can be used, such as, e.g., LIPOFECTIN®(including DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECTAMINE®(including DOSPA and DOPE, available from GIBCO/BRL). KL10, KL22, andKL25 are described, for example, in U.S. Pat. No. 8,691,750, which isincorporated herein by reference in its entirety. In particularembodiments, the lipid is DLin-MC3-DMA or DLin-KC2-DMA.

Anionic lipids suitable for use in lipid nanoparticles of the disclosureinclude, but are not limited to, phosphatidylglycerol, cardiolipin,diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanoloamine, N-succinyl phosphatidylethanolamine,N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, andother anionic modifying groups joined to neutral lipids.

Neutral lipids suitable for use in lipid nanoparticles of the disclosureinclude, but are not limited to, diacylphosphatidylcholine,diacylphosphatidylethanolamine, ceramide, sphingomyelin,dihydrosphingomyelin, cephalin, and cerebrosides. Lipids having avariety of acyl chain groups of varying chain length and degree ofsaturation are available or may be isolated or synthesized by well-knowntechniques. Additionally, lipids having mixtures of saturated andunsaturated fatty acid chains can be used. In some embodiments, theneutral lipids used in the disclosure are DOPE, DSPC, DPPC, POPC, or anyrelated phosphatidylcholine. In some embodiments, the neutral lipid maybe composed of sphingomyelin, dihydrosphingomyeline, or phospholipidswith other head groups, such as serine and inositol.

In some embodiments, amphipathic lipids are included in nanoparticles ofthe disclosure. Exemplary amphipathic lipids suitable for use innanoparticles of the disclosure include, but are not limited to,sphingolipids, phospholipids, and aminolipids. In some embodiments, aphospholipid is selected from the group consisting of1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine(OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine,1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphoetha nolamine (DOPE),1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG),and sphingomyelin. Other phosphorus-lacking compounds, such assphingolipids, glycosphingolipid families, diacylglycerols, andβ-acyloxyacids, may also be used. Additionally, such amphipathic lipidscan be readily mixed with other lipids, such as triglycerides andsterols.

In some embodiments, the lipid component of a nanoparticle of thedisclosure may include one or more PEGylated lipids. A PEGylated lipid(also known as a PEG lipid or a PEG-modified lipid) is a lipid modifiedwith polyethylene glycol. The lipid component may include one or morePEGylated lipids. A PEGylated lipid may be selected from thenon-limiting group consisting of PEG-modified phosphatidylethanolamines,PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkyl amines, PEG-modified diacylglycerols, and PEG-modifieddialkylglycerols. For example, a PEGylated lipid may be PEG-c-DOMG,PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

A lipid nanoparticle of the disclosure may include one or morestructural lipids. Exemplary, non-limiting structural lipids that may bepresent in the lipid nanoparticles of the disclosure includecholesterol, fecosterol, sitosterol, campesterol, stigmasterol,brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, oralpha-tocopherol).

In some embodiments, one or more mRNAs, of the disclosure may beformulated in a lipid nanoparticle having a diameter from about 1 nm toabout 900 nm, e.g., about 1 nm to about 100 nm, about 1 nm to about 200nm, about 1 nm to about 300 nm, about 1 nm to about 400 nm, about 1 nmto about 500 nm, about 1 nm to about 600 nm, about 1 nm to about 700 nm,about 1 nm to 800 nm, about 1 nm to about 900 nm. In some embodiments,the nanoparticle may have a diameter from about 10 nm to about 300 nm,about 20 nm to about 200 nm, about 30 nm to about 100 nm, or about 40 nmto about 80 nm. In some embodiments, the nanoparticle may have adiameter from about 30 nm to about 300 nm, about 40 nm to about 200 nm,about 50 nm to about 150 nm, about 70 to about 110 nm, or about 80 nm toabout 120 nm. In one embodiment, an mRNA, may be formulated in a lipidnanoparticle having a diameter from about 10 to about 100 nm includingranges in between such as, but not limited to, about 10 to about 20 nm,about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 toabout 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm,about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 toabout 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50to about 60 nm, about 50 to about 70 nm about 50 to about 80 nm, about50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm,about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about100 nm, about 80 to about 90 nm, about 80 to about 100 nm, and/or about90 to about 100 nm. In one embodiment, an mRNA may be formulated in alipid nanoparticle having a diameter from about 30 nm to about 300 nm,about 40 nm to about 200 nm, about 50 nm to about 150 nm, about 70 toabout 110 nm, or about 80 nm to about 120 nm including ranges inbetween.

In some embodiments, a lipid nanoparticle may have a diameter greaterthan 100 nm, greater than 150 nm, greater than 200 nm, greater than 250nm, greater than 300 nm, greater than 350 nm, greater than 400 nm,greater than 450 nm, greater than 500 nm, greater than 550 nm, greaterthan 600 nm, greater than 650 nm, greater than 700 nm, greater than 750nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, orgreater than 950 nm.

In some embodiments, the particle size of the lipid nanoparticle may beincreased and/or decreased. The change in particle size may be able tohelp counter a biological reaction such as, but not limited to,inflammation, or may increase the biological effect of the mRNA,delivered to a patient or subject.

In certain embodiments, it is desirable to target a nanoparticle, e.g.,a lipid nanoparticle, of the disclosure using a targeting moiety that isspecific to a cell type and/or tissue type. In some embodiments, ananoparticle may be targeted to a particular cell, tissue, and/or organusing a targeting moiety. In particular embodiments, a nanoparticlecomprises one or more mRNA described herein and a targeting moiety.Exemplary non-limiting targeting moieties include ligands, cell surfacereceptors, glycoproteins, vitamins (e.g., riboflavin) and antibodies(e.g., full-length antibodies, antibody fragments (e.g., Fv fragments,single chain Fv (scFv) fragments, Fab′ fragments, or F(ab′)2 fragments),single domain antibodies, camelid antibodies and fragments thereof,human antibodies and fragments thereof, monoclonal antibodies, andmultispecific antibodies (e.g., bispecific antibodies)). In someembodiments, the targeting moiety may be a polypeptide. The targetingmoiety may include the entire polypeptide (e.g., peptide or protein) orfragments thereof. A targeting moiety is typically positioned on theouter surface of the nanoparticle in such a manner that the targetingmoiety is available for interaction with the target, for example, a cellsurface receptor. A variety of different targeting moieties and methodsare known and available in the art, including those described, e.g., inSapra et al., Prog. Lipid Res. 42(5):439-62, 2003 and Abra et al., J.Liposome Res. 12:1-3, 2002.

In some embodiments, a lipid nanoparticle (e.g., a liposome) may includea surface coating of hydrophilic polymer chains, such as polyethyleneglycol (PEG) chains (see, e.g., Allen et al., Biochimica et BiophysicaActa 1237: 99-108, 1995; DeFrees et al., Journal of the AmericanChemistry Society 118: 6101-6104, 1996; Blume et al., Biochimica etBiophysica Acta 1149: 180-184, 1993; Klibanov et al., Journal ofLiposome Research 2: 321-334, 1992; U.S. Pat. No. 5,013,556; Zalipsky,Bioconjugate Chemistry 4: 296-299, 1993; Zalipsky, FEBS Letters 353:71-74, 1994; Zalipsky, in Stealth Liposomes Chapter 9 (Lasic and Martin,Eds) CRC Press, Boca Raton Fla., 1995. In one approach, a targetingmoiety for targeting the lipid nanoparticle is linked to the polar headgroup of lipids forming the nanoparticle. In another approach, thetargeting moiety is attached to the distal ends of the PEG chainsforming the hydrophilic polymer coating (see, e.g., Klibanov et al.,Journal of Liposome Research 2: 321-334, 1992; Kirpotin et al., FEBSLetters 388: 115-118, 1996).

Standard methods for coupling the targeting moiety or moieties may beused. For example, phosphatidylethanolamine, which can be activated forattachment of targeting moieties, or derivatized lipophilic compounds,such as lipid-derivatized bleomycin, can be used. Antibody-targetedliposomes can be constructed using, for instance, liposomes thatincorporate protein A (see, e.g., Renneisen et al., J. Bio. Chem.,265:16337-16342, 1990 and Leonetti et al., Proc. Natl. Acad. Sci. (USA),87:2448-2451, 1990). Other examples of antibody conjugation aredisclosed in U.S. Pat. No. 6,027,726. Examples of targeting moieties canalso include other polypeptides that are specific to cellularcomponents, including antigens associated with neoplasms or tumors.Polypeptides used as targeting moieties can be attached to the liposomesvia covalent bonds (see, for example Heath, Covalent Attachment ofProteins to Liposomes, 149 Methods in Enzymology 111-119 (AcademicPress, Inc. 1987)). Other targeting methods include the biotin-avidinsystem.

In some embodiments, a lipid nanoparticle of the disclosure includes atargeting moiety that targets the lipid nanoparticle to a cellincluding, but not limited to, hepatocytes, colon cells, epithelialcells, hematopoietic cells, epithelial cells, endothelial cells, lungcells, bone cells, stem cells, mesenchymal cells, neural cells, cardiaccells, adipocytes, vascular smooth muscle cells, cardiomyocytes,skeletal muscle cells, beta cells, pituitary cells, synovial liningcells, ovarian cells, testicular cells, fibroblasts, B cells, T cells,reticulocytes, leukocytes, granulocytes, and tumor cells (includingprimary tumor cells and metastatic tumor cells). In particularembodiments, the targeting moiety targets the lipid nanoparticle to ahepatocyte. In other embodiments, the targeting moiety targets the lipidnanoparticle to a colon cell. In some embodiments, the targeting moietytargets the lipid nanoparticle to a liver cancer cell (e.g., ahepatocellular carcinoma cell) or a colorectal cancer cell (e.g., aprimary tumor or a metastasis).

Pharmaceutical Compositions

The present disclosure includes pharmaceutical compositions comprisingan mRNA or a nanoparticle (e.g., a lipid nanoparticle) described herein,in combination with one or more pharmaceutically acceptable excipient,carrier or diluent. In particular embodiments, the mRNA, is present in ananoparticle, e.g., a lipid nanoparticle. In particular embodiments, themRNA or nanoparticle is present in a pharmaceutical composition. Invarious embodiments, the mRNA, present in the pharmaceutical compositionis encapsulated in a nanoparticle, e.g., a lipid nanoparticle.

Pharmaceutical compositions may optionally include one or moreadditional active substances, for example, therapeutically and/orprophylactically active substances. Pharmaceutical compositions of thepresent disclosure may be sterile and/or pyrogen-free. Generalconsiderations in the formulation and/or manufacture of pharmaceuticalagents may be found, for example, in Remington: The Science and Practiceof Pharmacy 21^(st) ed., Lippincott Williams & Wilkins, 2005(incorporated herein by reference in its entirety). In particularembodiments, a pharmaceutical composition comprises an mRNA and a lipidnanoparticle, or complexes thereof.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, dividing, shaping and/or packaging the product into a desiredsingle- or multi-dose unit.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the disclosure will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may includebetween 0.1% and 100%, e.g., between 0.5% and 70%, between 1% and 30%,between 5% and 80%, or at least 80% (w/w) active ingredient. In someembodiments, the active agent is an mRNA encoding a protein of interestand at least one microRNA binding site for a miR expressed in immunecells, such as a miR-142-3p binding site.

The mRNAs, of the disclosure can be formulated using one or moreexcipients to: (1) increase stability; (2) increase cell transfection;(3) permit the sustained or delayed release (e.g., from a depotformulation of the mRNA); (4) alter the biodistribution (e.g., targetthe mRNA to specific tissues or cell types); (5) increase thetranslation of a polypeptide encoded by the mmRNA in vivo; and/or (6)alter the release profile of a polypeptide encoded by the mRNA in vivo.In addition to traditional excipients such as any and all solvents,dispersion media, diluents, or other liquid vehicles, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, excipients of the present disclosurecan include, without limitation, lipidoids, liposomes, lipidnanoparticles (e.g., liposomes and micelles), polymers, lipoplexes,core-shell nanoparticles, peptides, proteins, carbohydrates, cellstransfected with mRNAs (e.g., for transplantation into a subject),hyaluronidase, nanoparticle mimics and combinations thereof.Accordingly, the formulations of the disclosure can include one or moreexcipients, each in an amount that together increases the stability ofthe mRNA, increases cell transfection by the mRNA, increases theexpression of a polypeptide encoded by the mRNA, and/or alters therelease profile of a mRNA-encoded polypeptide. Further, the mRNAs of thepresent disclosure may be formulated using self-assembled nucleic acidnanoparticles.

Various excipients for formulating pharmaceutical compositions andtechniques for preparing the composition are known in the art (seeRemington: The Science and Practice of Pharmacy, 21st Edition, A. R.Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006;incorporated herein by reference in its entirety). The use of aconventional excipient medium may be contemplated within the scope ofthe present disclosure, except insofar as any conventional excipientmedium may be incompatible with a substance or its derivatives, such asby producing any undesirable biological effect or otherwise interactingin a deleterious manner with any other component(s) of thepharmaceutical composition. Excipients may include, for example:antiadherents, antioxidants, binders, coatings, compression aids,disintegrants, dyes (colors), emollients, emulsifiers, fillers(diluents), film formers or coatings, glidants (flow enhancers),lubricants, preservatives, printing inks, sorbents, suspensing ordispersing agents, sweeteners, and waters of hydration. Exemplaryexcipients include, but are not limited to: butylated hydroxytoluene(BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate,croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid,crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate,maltitol, mannitol, methionine, methylcellulose, methyl paraben,microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone,povidone, pregelatinized starch, propyl paraben, retinyl palmitate,shellac, silicon dioxide, sodium carboxymethyl cellulose, sodiumcitrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid,sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, andxylitol.

In some embodiments, the formulations described herein may include atleast one pharmaceutically acceptable salt. Examples of pharmaceuticallyacceptable salts that may be included in a formulation of the disclosureinclude, but are not limited to, acid addition salts, alkali or alkalineearth metal salts, mineral or organic acid salts of basic residues suchas amines; alkali or organic salts of acidic residues such as carboxylicacids; and the like. Representative acid addition salts include acetate,acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzene sulfonic acid, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like, aswell as nontoxic ammonium, quaternary ammonium, and amine cations,including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like.

In some embodiments, the formulations described herein may contain atleast one type of polynucleotide. As a non-limiting example, theformulations may contain 1, 2, 3, 4, 5 or more than 5 mRNAs describedherein.

Liquid dosage forms for e.g., parenteral administration include, but arenot limited to, pharmaceutically acceptable emulsions, microemulsions,nanoemulsions, solutions, suspensions, syrups, and/or elixirs. Inaddition to active ingredients, liquid dosage forms may comprise inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, oral compositions can includeadjuvants such as wetting agents, emulsifying and/or suspending agents.In certain embodiments for parenteral administration, compositions aremixed with solubilizing agents such as CREMAPHOR®, alcohols, oils,modified oils, glycols, polysorbates, cyclodextrins, polymers, and/orcombinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations may be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables. Injectableformulations can be sterilized, for example, by filtration through abacterial-retaining filter, and/or by incorporating sterilizing agentsin the form of sterile solid compositions which can be dissolved ordispersed in sterile water or other sterile injectable medium prior touse.

In some embodiments, pharmaceutical compositions including at least onemRNA described herein are administered to mammals (e.g., humans).Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions that aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to any other animal, e.g., to a non-human mammal.Modification of pharmaceutical compositions suitable for administrationto humans in order to render the compositions suitable foradministration to various animals is well understood, and the ordinarilyskilled veterinary pharmacologist can design and/or perform suchmodification with merely ordinary, if any, experimentation. Subjects towhich administration of the pharmaceutical compositions is contemplatedinclude, but are not limited to, humans and/or other primates; mammals,including commercially relevant mammals such as cattle, pigs, horses,sheep, cats, dogs, mice, and/or rats; and/or birds, includingcommercially relevant birds such as poultry, chickens, ducks, geese,and/or turkeys. In particular embodiments, a subject is provided withtwo or more mRNAs described herein, e.g., a first mRNA encoding a firstpolypeptide of interest and comprising at least one microRNA bindingsite for a miR expressed in immune cells and a second mRNA encoding asecond polypeptide of interest and comprising at least one microRNAbinding site for a miR expressed in immune cells. In particularembodiments, the first and second mmRNAs are provided to the subject atthe same time or at different times, e.g., sequentially. In particularembodiments, the first and second mRNAs are provided to the subject inthe same pharmaceutical composition or formulation, e.g., to facilitateuptake of both mRNAs by the same cells.

Inhibition of Immune Cell Activation and Cytokine Production

The methods of the disclosure allow for reducing or inhibiting unwantedimmune cell activation and/or unwanted cytokine production in a subjectbeing treated with an mRNA-based therapeutic, such as unwanted immunecell activation and/or cytokine production that is stimulated by apolypeptide of interest (e.g., therapeutic agent) encoded by themRNA-based therapeutic, by inclusion of at least one miR-126 (e.g.,miR-126-3p) and/or miR-142 (e.g., miR-142-3p) binding site in the mRNAconstruct. In one embodiment, the immune cell activation is lymphocyteactivation. In one embodiment, the immune cell activation is B cellactivation. In another embodiment, the immune cell activation is T cellactivation. In yet other embodiments, the immune cell activation ismacrophage activation, dendritic cell activation, NK cell activation,basophil activation or eosinophil activation.

In one embodiment, reduction or inhibition of unwanted immune cellactivation is determined compared to control administration of an mmRNA,lacking the at least one miR-126 or miR-142 microRNA binding site. Invarious embodiments, the immune cell activation is decreased by at least5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%,by at least 30%, by at least 35%, by at least 40%, by at least 45%, byat least 50%, by at least about 60%, by at least about 70%, by at leastabout 80%, by about 5%-50%, by about 10%-50%, by about 15%-50%, by about20%-50%, by about 25%-50%, by about 10%-80%, by about 10%-70%, by about10%-60%, by about 20%-80%, by about 20%-70%, by about 20%-60%, by about20%-40% or by about 25%-75%.

The level of immune cell activation can be evaluated by essentially anymethod established in the art for assessing immune cell activation, suchas the frequency of an activated immune cell population, typicallyassessed by detection of cells expressing cell-surface activationmarkers, or levels of production of one or more cytokines indicative ofimmune cell activation. In one embodiment, the immune cell activation isB cell activation, wherein the level of B cell activation is determinedby measuring the frequency of activated B cells, such as the frequencyof activated B cells among the splenic B cell population. B cell surfacemarkers indicative of B cell activation are well known in the art (seee.g., Maddalay, R. et al. (2010) FEBS Letters 584:4883-4894). In oneembodiment, B cell activation is determined by frequency of CD19⁺ CD86⁺CD69⁺B cells. In another embodiment, the immune cell activation is Bcell activation, wherein the level of B cell activation is determined bycytokine secretion, such as by secretion of interleukin-6 (IL-6), tumornecrosis factor α (TNF-α) or interferon-γ (IFN-γ), e.g., in the serum oftreated subjects. In one embodiment, B cell activation is determined bysecretion of IL-6, e.g., in the serum of treated subjects. In otherembodiments, the unwanted cytokine production that is reduced orinhibited is production of interleukin-6 (IL-6), tumor necrosis factor α(TNF-α) or interferon-γ (IFN-γ), e.g., in the serum of treated subjects.In another embodiment, the unwanted cytokine production that is reducedor inhibited is production of interleukin-6 (IL-6).

In one embodiment, the immune cell activation is plasmacytoid dendriticcell (pDC) activation, wherein the level of pDC activation is determinedby measuring the frequency of activated pDC, such as the frequency ofactivated pDCs among the splenic pDC population. pDC surface markersindicative of activation are well known in the art (see e.g., Dzionek,A., et al., (2002) Hum Immunol. Vol. 63(12): 1133-48). In oneembodiment, pDC activation is determined by frequency of CD11c⁺ CD70⁺CD86⁺ cells.

In one embodiment, unwanted immune cell activation and/or unwantedcytokine production is decreased without a corresponding decrease inexpression of a polypeptide of interest encoded by the mmRNA. Thus, themethods of the disclosure allow for inhibiting or reducing immune cellactivation (e.g., B cell activation, cytokine production) in a subjecttreated with an mRNA encoding a polypeptide of interest that is atherapeutic agent without significantly affecting the level ofexpression of the therapeutic agent in the subject.

A standard metric that can be used in the methods of the disclosure isthe measure of the ratio of the level or amount of encoded polypeptide(protein) produced in a cell, tissue or organism to the level or amountof one or more (or a panel) of cytokines whose expression is triggeredin the cell, tissue or organism as a result of administration or contactwith the chemically modified mRNA. Such ratios are referred to herein asthe Protein: Cytokine Ratio or “PC” Ratio. The higher the PC ratio, themore efficacious the chemically modified mRNA (polynucleotide encodingthe protein measured). Preferred PC Ratios, by cytokine, of the presentdisclosure may be greater than 1, greater than 10, greater than 100,greater than 1000, greater than 10,000 or more. Modified mRNAs havinghigher PC Ratios than a modified mRNA of a different or unmodifiedconstruct are preferred.

The PC ratio may be further qualified by the percent modificationpresent in the mRNA. For example, normalized to a 100% modified mRNA,the protein production as a function of cytokine (or risk) or cytokineprofile can be determined.

In one embodiment, the present disclosure provides a method fordetermining, across chemistries, cytokines or percent modification, therelative efficacy of any particular modified mRNA comprising at leastone miR-126 and/or miR-142 binding site by comparing the PC Ratio of themodified mRNA including the miR-126 and/or miR-142 binding site(s) tothe PC ratio of the same construct without the miR-126 and/or miR-142binding site(s).

In one embodiment, the level of expression of a polypeptide of interestencoded by the mmRNA in the serum of a mammal (e.g., human) can be atleast 50 pg/ml at least two hours after administration. In anotherembodiment, the level of expression of a polypeptide of interest encodedby the mmRNA in the serum of a mammal (e.g., human) can remain above 50pg/ml for at least 72 hours after administration. In another embodiment,the level of expression of a polypeptide of interest encoded by themmRNA in the serum of a mammal (e.g., human) can remain above 60 pg/mlfor at least 72 hours after administration.

Inhibition of B1a Cells/Inhibition of Accelerated Blood Clearance

The spleen has been previously implicated in ABC, although the precisemechanism(s) responsible for ABC have not previously beeen understood.The spleen is composed of red pulp (red blood cell-rich), white pulp(lymphocyte-rich), and the marginal zone (located between the red andwhite pulp and outside the marginal sinus). Antigens entering the spleenare retained in the marginal zone, where blood-flow is reduced to allowinteraction between antigens and immune effector cells (e.g., B cells)(Harms et al. Infect. Immuno. Vol. 64: 4220-4225, 1996). The spleen'srole in accelerated blood clearance is thought to be significant.Biodistribution data demonstrates that lipid-comprising compounds orcompositions (e.g., LNPs) are taken up by the spleen (data not shown).Histological evaluation shows uptake of LNPs in the marginal zonerapidly after dosing (e.g., IV dosing). Within the spleen, LNPs caninteract with splenic B cells, contributing to various elements of theimmune response to the LNPs. For example, certain components of the LNPs(e.g., PEG components) can interact with CD19+ B cells in the spleen,resulting in binding, internalization, membrane fusion, and/oractivation of such cells, resulting in production of IgG and/or IgMmolecules specific for components of the LNPs, leading to acceleratedblood clearance, for example, on second or subsequent dosing with theLNPs.

Suprisingly, it has also been demonstrated herein that particular cellsof the immune system, namely, pDCs, also contribute to the ABCphenomenon. It has been demonstrated herein that inclusion of miRbinding sites (e.g., miR-126 binding sites) can lead to reduction ofunwanted immune responses (e.g., ADA) against proteins encoded byLNP-encapsulated mRNAs, for example, modified mRNAs. It has now alsobeen surprisingly demonstrated that inclusion of miR binding sites, inparticular, miR-126 binding sites, can lead to reduction in furtherunwanted immune responses against LNPs encapsulating the mRNAs. miR-126(e.g., miR-126-3p) is highly expressed in pDCs and is, in fact,upregulated during activation of pDCs. pDCs synergistically increase Bcell activation in response to nucleic acids and other forms ofactivation via cytokine secretion and plasma cell activation. Moreover,“miR-126-3p low” pDCs (e.g., pDC in which miR-126 has been knocked downor knocked out) are activation impaired (e.g., unable to launch aneffective immune response to nuleic acids, secrete IFN-α/β secretion,IL-6 secretion, etc., inability to migrate to spleen upon activation andthe like). As demonstrated herein, inclusion of miR-126 binding sites inmRNAs results in low B cell activation and low serum IL-6 over severalweeks of repeat dosing of LNP-encapsulated mRNAs. Protein expression ismaintained over similar dosing schedules. Suprisingly, anti-PEG IgMresponses are dramatically diminished over weeks of repeat dosing. Thus,an unexpected benefit of inclusion of miR-126 binding sites in mRNA, inparticular, in LNP-encapsulated mRNA, is reduction of ABC.

In the spleen, for example, in the marginal zone of the spleen, certainif these key immune cells can interact either directly or indirectly,e.g., as a result of cytokine production (e.g., IL-6).

Without wishing to be bound by theory, this disclosure provides evidencethat miRs expressed in immune cells present in the marginal zoneparticipate in accelerated blood clearance. When an mRNA of thedisclosure includes one or more miR binding sites that bind to one ormore miRs expressed in immune cells, the miR of interest isdownregulated (e.g., antagonized and/or degraded). The inclusion of atleast one miR binding site that binds to at least one miR expressed inimmune cells results in decreased production of IgM molecules capable ofbinding lipid components (e.g., PEG lipids), compared to mRNA withoutthe at least one miR binding site. Given the known role of IgM moleculesin accelerated blood clearance, the ability of a miR binding site thatbinds a miR expressed in immune cells to inhibit or reduce production ofIgM molecules, indicates an important role of miRs expressed in immunecells, specifically in the marginal zone of the spleen, in acceleratedblood clearance.

The methods of the disclosure allow for reducing or inhibitingaccelerated blood clearance in a subject repeatedly administered amessenger RNA (mRNA) encoding a polypeptide of interest encapsulated inan lipid nanoparticle (LNP), the method comprising administering to thesubject a chemically modified mRNA encoding the polypeptide of interestencapsulated in an lipid nanoparticle (LNP), wherein the chemicallymodified mRNA comprises at least one microRNA binding site for amicroRNA expressed in immune cells, and wherein the chemically modifiedmRNA comprises one or more modified nucleobases, such that acceleratedblood clearance is reduced or inhibited in the subject upon repeatadministration.

In other embodiments, accelerated blood clearance is reduced orinhibited in a subject administered a messenger RNA (mRNA) encoding apolypeptide of interest encapsulated in an lipid nanoparticle (LNP), byadministering to the subject intravenously a first dose of a chemicallymodified mRNA encapsulated in an lipid nanoparticle (LNP), wherein thechemically modified mRNA comprises at least one microRNA binding sitefor a microRNA expressed in immune cells, and wherein the chemicallymodified mRNA comprises one or more modified nucleobases; andadministering to the subject intravenously a second dose of thechemically modified mRNA encapsulated in an LNP, such that acceleratedblood clearance is reduced or inhibited in the subject.

In some embodiments, accelerated blood clearance is reduced or inhibitedin a subject administered multiple doses of a messenger RNA (mRNA)encoding a polypeptide of interest encapsulated in an lipid nanoparticle(LNP), by administering to the subject a chemically modified mRNAencoding the polypeptide of interest encapsulated in an lipidnanoparticle (LNP), wherein the chemically modified mRNA comprises atleast one microRNA binding site for a microRNA expressed in immunecells, and wherein the chemically modified mRNA comprises one or moremodified nucleobases, such that accelerated blood clearance is reducedor inhibited in the subject upon administration of one or moresubsequent doses.

In other embodiments, accelerated blood clearance is reduced or inhbitedin a subject administered a messenger RNA (mRNA) encoding a polypeptideof interest encapsulated in an lipid nanoparticle (LNP), byadministering to the subject a chemically modified mRNA encoding thepolypeptide of interest encapsulated in an lipid nanoparticle (LNP),wherein the chemically modified mRNA comprises at least one microRNAbinding site for a microRNA expressed in immune cells, and wherein thechemically modified mRNA comprises one or more modified nucleobases,such that accelerated blood clearance is reduced or inhibited in thesubject upon administration of a subsequent dose of the mRNA.

In further embodiments, accelerated blood clearance is reduced orinhibited in a subject repeatedly administered a messenger RNA (mRNA)encoding a polypeptide of interest encapsulated in an lipid nanoparticle(LNP), by administering to the subject a chemically modified mRNAencoding the polypeptide of interest encapsulated in an lipidnanoparticle (LNP), wherein the chemically modified mRNA comprises atleast one microRNA binding site for a microRNA expressed in immunecells, wherein the chemically modified mRNA comprises one or moremodified nucleobases, and wherein the LNP does not activate B cellsand/or does not induce production of IgM molecules capable of binding tothe LNP, such that accelerated blood clearance is reduced or inhibitedin the subject upon repeat administration.

In further embodiments, accelerated blood clearance is reduced orinhibited in a subject administered a messenger RNA (mRNA) encoding apolypeptide of interest encapsulated in an lipid nanoparticle (LNP), byadministering to the subject intravenously a first dose of a chemicallymodified mRNA encapsulated in an lipid nanoparticle (LNP), wherein thechemically modified mRNA comprises at least one microRNA binding sitefor a microRNA expressed in immune cells, and wherein the chemicallymodified mRNA comprises one or more modified nucleobases; andadministering to the subject intravenously a second dose of thechemically modified mRNA encapsulated in an LNP, wherein the LNP doesnot activate B cells and/or does not induce production of IgM moleculescapable of binding to the LNP, such that accelerated blood clearance isreduced or inhibited in the subject.

In other embodiments, accelerated blood clearance is reduced orinhibited in a subject administered multiple doses of a messenger RNA(mRNA) encoding a polypeptide of interest encapsulated in an lipidnanoparticle (LNP), by administering to the subject a chemicallymodified mRNA encoding the polypeptide of interest encapsulated in anlipid nanoparticle (LNP), wherein the chemically modified mRNA comprisesat least one microRNA binding site for a microRNA expressed in immunecells, wherein the chemically modified mRNA comprises one or moremodified nucleobases, and wherein the LNP does not activate B cellsand/or does not induce production of IgM molecules capable of binding tothe LNP, such that accelerated blood clearance is reduced or inhibitedin the subject upon administration of one or more subsequent doses.

In some embodiments, accelerated blood clearance is reduced or inhibitedin a subject administered a messenger RNA (mRNA) encoding a polypeptideof interest encapsulated in an lipid nanoparticle (LNP), byadministering to the subject a chemically modified mRNA encoding thepolypeptide of interest encapsulated in an lipid nanoparticle (LNP),wherein the chemically modified mRNA comprises at least one microRNAbinding site for a microRNA expressed in immune cells, wherein thechemically modified mRNA comprises one or more modified nucleobases, andwherein the LNP does not activate B cells and/or does not induceproduction of IgM molecules capable of binding to the LNP, such thataccelerated blood clearance is reduced or inhibited in the subject uponadministration of a subsequent dose of the mRNA.

In some embodiments, the disclosure provides a method of reducing orinhibiting production of IgM molecules that recognize polyethyleneglycol (PEG) in a subject repeatedly administered a messenger RNA (mRNA)encoding a polypeptide of interest encapsulated in an lipid nanoparticle(LNP), by administering to the subject a chemically modified mRNAencoding the polypeptide of interest encapsulated in an lipidnanoparticle (LNP), wherein the chemically modified mRNA comprises atleast one microRNA binding site for a microRNA expressed in immunecells, and wherein the chemically modified mRNA comprises one or moremodified nucleobases, such that production of IgM molecules thatrecognize PEG are reduced or inhibited in the subject upon repeatadministration.

In some embodiments, production of IgM molecules that recognizepolyethylene glycol (PEG) is reduced or inhibited in a subjectadministered a messenger RNA (mRNA) encoding a polypeptide of interestencapsulated in an lipid nanoparticle (LNP), by administering to thesubject intravenously a first dose of a chemically modified mRNAencapsulated in an lipid nanoparticle (LNP), wherein the chemicallymodified mRNA comprises at least one microRNA binding site for amicroRNA expressed in immune cells, and wherein the chemically modifiedmRNA comprises one or more modified nucleobases; and administering tothe subject intravenously a second dose of the chemically modified mRNAencapsulated in an LNP, such that production of IgM molecules thatrecognize PEG are reduced or inhibited in the subject.

In some embodiments, production of IgM molecules that recognizepolyethylene glycol (PEG) is reduced or inhibited in a subjectadministered multiple doses of a messenger RNA (mRNA) encoding apolypeptide of interest encapsulated in an lipid nanoparticle (LNP), byadministering to the subject a chemically modified mRNA encoding thepolypeptide of interest encapsulated in an lipid nanoparticle (LNP),wherein the chemically modified mRNA comprises at least one microRNAbinding site for a microRNA expressed in immune cells, and wherein thechemically modified mRNA comprises one or more modified nucleobases,such that production of IgM molecules that recognize PEG are reduced orinhibited in the subject upon administration of one or more subsequentdoses.

In other embodiments, production of IgM molecules that recognizepolyethylene glycol (PEG) is reduced or inhibited in a subjectadministered a messenger RNA (mRNA) encoding a polypeptide of interestencapsulated in an lipid nanoparticle (LNP), by administering to thesubject a chemically modified mRNA encoding the polypeptide of interestencapsulated in an lipid nanoparticle (LNP), wherein the chemicallymodified mRNA comprises at least one microRNA binding site for amicroRNA expressed in immune cells, and wherein the chemically modifiedmRNA comprises one or more modified nucleobases, such that production ofIgM molecules that recognize PEG are reduced or inhibited in the subjectupon administration of a subsequent dose of the mRNA.

In further embodiments, the disclosure provides a method of reducing orinhibiting activation of B1a cells in a subject repeatedly administereda messenger RNA (mRNA) encoding a polypeptide of interest encapsulatedin an lipid nanoparticle (LNP), by comprising administering to thesubject a chemically modified mRNA encoding the polypeptide of interestencapsulated in an lipid nanoparticle (LNP), wherein the chemicallymodified mRNA comprises at least one microRNA binding site for amicroRNA expressed in immune cells, and wherein the chemically modifiedmRNA comprises one or more modified nucleobases, such that activation ofB1a cells is reduced or inhibited in the subject upon repeatadministration.

In some embodiments, activation of B1a cells is reduced or inhibited ina subject administered a messenger RNA (mRNA) encoding a polypeptide ofinterest encapsulated in an lipid nanoparticle (LNP), by administeringto the subject intravenously a first dose of a chemically modified mRNAencapsulated in an lipid nanoparticle (LNP), wherein the chemicallymodified mRNA comprises at least one microRNA binding site for amicroRNA expressed in immune cells, and wherein the chemically modifiedmRNA comprises one or more modified nucleobases; and administering tothe subject intravenously a second dose of the chemically modified mRNAencapsulated in an LNP, such that activation of B1a cells is reduced orinhibited in the subject.

In other embodiments, activation of B1a cells is reduced or inhibited ina subject administered multiple doses of a messenger RNA (mRNA) encodinga polypeptide of interest encapsulated in an lipid nanoparticle (LNP),by administering to the subject a chemically modified mRNA encoding thepolypeptide of interest encapsulated in an lipid nanoparticle (LNP),wherein the chemically modified mRNA comprises at least one microRNAbinding site for a microRNA expressed in immune cells, and wherein thechemically modified mRNA comprises one or more modified nucleobases,such that activation of B1a cells is reduced or inhibited in the subjectupon administration of one or more subsequent doses.

In some embodiments, activation of B1a cells is reduced or inhibited ina subject administered a messenger RNA (mRNA) encoding a polypeptide ofinterest encapsulated in an lipid nanoparticle (LNP), by administeringto the subject a chemically modified mRNA encoding the polypeptide ofinterest encapsulated in an lipid nanoparticle (LNP), wherein thechemically modified mRNA comprises at least one microRNA binding sitefor a microRNA expressed in immune cells, and wherein the chemicallymodified mRNA comprises one or more modified nucleobases, such thatactivation of B1a cells is reduced or inhibited in the subject uponadministration of a subsequent dose of the mRNA.

In further embodiments, the disclosure provides a method of reducing orinhibiting activation of plasmacytoid dendrtic cells in a subjectrepeatedly administered a messenger RNA (mRNA) encoding a polypeptide ofinterest encapsulated in an lipid nanoparticle (LNP), by administeringto the subject a chemically modified mRNA encoding the polypeptide ofinterest encapsulated in an lipid nanoparticle (LNP), wherein thechemically modified mRNA comprises at least one microRNA binding sitefor a microRNA expressed in immune cells, and wherein the chemicallymodified mRNA comprises one or more modified nucleobases, such thatactivation of plasmacytoid dendritic cells is reduced or inhibited inthe subject upon repeat administration.

In some embodiments, activation of plasmacytoid dendritic cells isreduced or inhibited in a subject administered a messenger RNA (mRNA)encoding a polypeptide of interest encapsulated in an lipid nanoparticle(LNP), by administering to the subject intravenously a first dose of achemically modified mRNA encapsulated in an lipid nanoparticle (LNP),wherein the chemically modified mRNA comprises at least one microRNAbinding site for a microRNA expressed in immune cells, and wherein thechemically modified mRNA comprises one or more modified nucleobases; andadministering to the subject intravenously a second dose of thechemically modified mRNA encapsulated in an LNP, such that activation ofplasmacytoid dendritic cells is reduced or inhibited in the subject.

In some embodiments, activation of plasmacytoid dendritic cells isreduced or inhibited in a subject administered multiple doses of amessenger RNA (mRNA) encoding a polypeptide of interest encapsulated inan lipid nanoparticle (LNP), by administering to the subject achemically modified mRNA encoding the polypeptide of interestencapsulated in an lipid nanoparticle (LNP), wherein the chemicallymodified mRNA comprises at least one microRNA binding site for amicroRNA expressed in immune cells, and wherein the chemically modifiedmRNA comprises one or more modified nucleobases, such that activation ofplasmacytoid dendritic cells is reduced or inhibited in the subject uponadministration of one or more subsequent doses.

In some embodiments, activation of plasmacytoid dendritic cells isreduced or inhibited in a subject administered a messenger RNA (mRNA)encoding a polypeptide of interest encapsulated in an lipid nanoparticle(LNP), by administering to the subject a chemically modified mRNAencoding the polypeptide of interest encapsulated in an lipidnanoparticle (LNP), wherein the chemically modified mRNA comprises atleast one microRNA binding site for a microRNA expressed in immunecells, and wherein the chemically modified mRNA comprises one or moremodified nucleobases, such that activation of plasmacytoid dendriticcells is reduced or inhibited in the subject upon administration of asubsequent dose of the mRNA.

In further embodiments, the mRNA encoding a polypeptide of interestencapsulated in a lipid nanoparticle (LNP) does not activate B cellsand/or does not induce production of IgM molecules capable of binding tothe LNP. In some embodiments the mRNA encoding a polypeptide of interestencapsulated in a lipid nanoparticle (LNP) does not activate B cells. Inother embodiments, the mRNA encoding a polypeptide of interestencapsulated in a lipid nanoparticle (LNP) does not induce production ofIgM molecules capable of binding to the LNP.

In some embodiments, reduction or inhibition of accelerated bloodclearance is determined compared to control administration of achemically modified mRNA lacking the at least one microRNA binding siteencapsulated in a lipid nanoparticle (LNP). In other embodiments,accelerated blood clearance is reduced or inhibited without acorresponding reduction or inhibition in expression of the polypeptideof interest encoded by the chemically modified mRNA.

In further embodiments, wherein the interval between two consecutivedoses is less than 2 weeks. In some embodiments, the interval betweentwo consecutive doses is less than 1 week.

In some emdodiments, the IgM molecules recognize polyethylene glycol(PEG).

Methods of the Disclosure

In one aspect, the disclosure pertains to a method of reducing orinhibiting an anti-drug antibody response in a subject, comprisingadministering to the subject a modified messenger RNA (mmRNA) encoding apolypeptide of interest, wherein the mmRNA comprises at least onemicroRNA binding site for a miR expressed in immune cells (e.g.,miR-142-3p and/or miR-126-3p), and wherein the mmRNA comprises one ormore modified nucleobases, such that an anti-drug antibody response tothe polypeptide of interest is reduced or inhibited in the subject. Asdescribed above, in various embodiments, the mmRNA can comprise, forexample, two or more, 1-4, one, two, three or four binding sites for oneor more miRs expressed in immune cells. In certain embodiments, themmRNA comprises at least two binding sites for at least two differentmiRs expressed in immune cells. For example, the mmRNA can comprise afirst binding site for miR-142-3p and a second binding site for adifferent miR expressed in an immune cell, such as miR-155, miR-146(miR-146-3p and/or miR-146-5p) or miR-126. Alternatively, the mmRNA cancomprise a first binding site for miR-126 (e.g., miR-126-3p) and asecond binding site for a different miR expressed in an immune cell,such as miR-142 (mir-142-3p and/or miR-142-5p), miR-155 or miR-146(miR-146-3p and/or miR-146-5p). In one embodiment, the mmRNA comprises afirst binding site for miR-142-3p and a second binding site for miR-126.

In related embodiments, the subject is provided with or administered ananoparticle (e.g., a lipid nanoparticle) comprising the mmRNA. Infurther related embodiments, the subject is provided with oradministered a pharmaceutical composition of the disclosure to thesubject. In particular embodiments, the pharmaceutical compositioncomprises an mmRNA encoding a polypeptide of interest and comprising atleast one miR binding site as described herein, or it comprises ananoparticle comprising the mmRNA. In particular embodiments, the mmRNAis present in a nanoparticle, e.g., a lipid nanoparticle. In particularembodiments, the mmRNA or nanoparticle is present in a pharmaceuticalcomposition.

In one embodiment, the mmRNA is administered intravenously encapsulatedin a lipid nanoparticle. In one embodiment, the mmRNA is administered byonce weekly infusion (e.g., intravenous infusion, such as via a pump).In one embodiment, the mmRNA is administered by once weekly infusion forat least 4 weeks.

In another embodiment, the disclosure provides a method of reducing orinhibiting an anti-drug antibody response following repeatedadministration of a polypeptide of interest to a subject, comprisingadministering to the subject intravenously a first dose of a modifiedmRNA (mmRNA) encoding a polypeptide of interest encapsulated in an LNP,wherein the mmRNA comprises at least one binding site for a miRexpressed in immune cells (e.g., a miR-142-3p microRNA binding siteand/or a miR-126 microRNA binding site), and wherein the mmRNA comprisesone or more modified nucleobases; and administering to the subjectintravenously a second dose of the mmRNA encapsulated in an LNP, suchthat an anti-drug antibody response to the polypeptide of interest isreduced or inhibited in the subject.

In another aspect, the disclosure provides a method of reducing orinhibiting an anti-drug antibody response following repeatedadministration of a polypeptide of interest to a subject, comprising

(i) administering to the subject intravenously a first dose of amodified mRNA (mmRNA) encoding a polypeptide of interest encapsulated inan LNP, wherein the mmRNA comprises at least one microRNA binding sitefor a miR expressed in immune cells (e.g., a miR-142-3p microRNA bindingsite and/or a miR-126 microRNA binding site), and wherein the mmRNAcomprises one or more modified nucleobases;

(ii) detecting a level of anti-drug antibodies in a sample from thesubject; and

(iii) administering to the subject intravenously a second dose of themmRNA encapsulated in an LNP when the level of anti-drug antibodies inthe sample is diminished, such that an anti-drug antibody response tothe polypeptide of interest is reduced or inhibited in the subject.

Given the ability of the methods of the disclosure to reduce or inhibitexpression of the protein of interest encoded by the mmRNA in the spleenof the subject to which the mmRNA is administered, the disclosurefurther provides methods for reducing toxicity of mmRNA-basedtherapeutics. Accordingly, in another aspect, the disclosure provides amethod of reducing or inhibiting drug-related toxicity in a subject,comprising administering to the subject a modified messenger RNA (mmRNA)encoding a polypeptide of interest, wherein the mmRNA comprises at leastone binding site for a miR expressed in immune cells (e.g., a miR-142-3pmicroRNA binding site and/or a miR-126 microRNA binding site), andwherein the mmRNA comprises one or more modified nucleobases, such thatdrug-related toxicity to the polypeptide of interest is reduced orinhibited in the subject. In one embodiment, the drug-related toxicityto the polypeptide of interest is decreased blood cell counts(cytopenia) in the subject. In one embodiment, the drug-related toxicityto the polypeptide of interest is autoimmunity in the subject. In oneembodiment, the drug-related toxicity to the polypeptide of interest iscomplement-mediated effects in the subject. In one embodiment, thedrug-related toxicity to the polypeptide of interest is decreasedhematopoiesis in the subject. In other embodiments, the drug-relatedtoxicity can be, for example, renal toxicity or liver toxicity.

In another aspect, the disclosure pertains to a method of reducing orinhibiting unwanted immune cell activation in a subject administered anRNA, e.g., a messenger RNA (mRNA), comprising administering to thesubject an RNA, e.g., a mRNA (e.g., a chemically modified mRNA ormmRNA), wherein the mRNA, e.g., chemically modified RNA or mmRNA,comprises at least one miR-126 and/or miR-142 microRNA binding site, andwherein the mRNA, e.g., chemically modified mRNA or mmRNA, comprises oneor more modified nucleobases, such that unwanted immune cell activationis reduced or inhibited in the subject. In another aspect, thedisclosure pertains to a method of reducing or inhibiting unwantedcytokine production in a subject administered an RNA, e.g., a messengerRNA (mRNA), the method comprising administering to the subject an RNA,e.g., a mRNA (e.g., a chemically modified mRNA or mmRNA), wherein themRNA, e.g., chemically modified mRNA or mmRNA, comprises at least onemiR-126 and/or miR-142 microRNA binding site, and wherein the mRNA,e.g., chemically modified mmRNA comprises one or more modifiednucleobases, such that unwanted cytokine production is reduced orinhibited in the subject.

As described above, in various embodiments, the chemically modified mRNA(referred to as mmRNA) can comprise, for example, two or more, 1-4, one,two, three or four binding sites for one or more miRs expressed inimmune cells. In certain embodiments, the mmRNA, comprises at least twobinding sites for at least two different miRs expressed in immune cells.For example, the mmRNA, can comprise a first binding site for miR-126and a second binding site for a different miR expressed in an immunecell, such as miR-142 (miR-142-3p and/or miR-142-5p), miR-155 or miR-146(miR-146-3p and/or miR-146-5p). Alternatively, the mmRNA, can comprise afirst binding site for miR-142 (miR-142-3p and/or miR-142-5p) and asecond binding site for a different miR expressed in an immune cell,such as miR-126, miR-155 or miR-146 (miR-146-3p and/or miR-146-5p). Inone embodiment, the mmRNA comprises a first binding site for miR-142-3pand a second binding site for miR-126.

In certain embodiments, the mRNA encodes a polypeptide of interest(e.g., a therapeutic agent), wherein unwanted immune cell activationoccurs in response to the polypeptide of interest.

In related embodiments, the subject is provided with or administered ananoparticle (e.g., a lipid nanoparticle) comprising the mRNA, e.g.,mmRNA. In further related embodiments, the subject is provided with oradministered a pharmaceutical composition of the disclosure to thesubject. In particular embodiments, the pharmaceutical compositioncomprises an mmRNA encoding a polypeptide of interest and comprising atleast one miR binding site as described herein, or it comprises ananoparticle comprising the mmRNA. In particular embodiments, the mmRNAis present in a nanoparticle, e.g., a lipid nanoparticle. In particularembodiments, the mmRNA or nanoparticle is present in a pharmaceuticalcomposition.

In one embodiment, the mRNA, e.g., mmRNA is administered intravenouslyencapsulated in a lipid nanoparticle. In one embodiment, the mRNA, e.g.,mmRNA is administered by once weekly infusion (e.g., intravenousinfusion, such as via a pump). In one embodiment, the mRNA, e.g., mmRNAis administered by once weekly infusion for at least 4 weeks.

In another embodiment, the disclosure provides a method of reducing orinhibiting unwanted immune cell activation (e.g., lymphocyte activation,B cell activation) or unwanted cytokine production in a subjectadministered a messenger RNA (mRNA), the method comprising administeringto the subject intravenously a first dose of a mRNA, e.g., chemicallymodified mRNA (mmRNA) encapsulated in an LNP, wherein the mRNA, e.g.,mmRNA comprises at least one miR-126 and/or miR-142 microRNA bindingsite, and wherein the mRNA, eg., mmRNA, comprises one or more modifiednucleobases; and administering to the subject intravenously a seconddose of the mRNA, e.g., mmRNA, encapsulated in an LNP, such thatunwanted immune cell activation or unwanted cytokine production isreduced or inhibited in the subject.

In certain embodiments, the mRNA encodes a polypeptide of interest(e.g., a therapeutic agent), wherein unwanted immune cell activationand/or unwanted cytokine production occurs in response to thepolypeptide of interest.

In another aspect, the disclosure provides a method of reducing orinhibiting unwanted immune cell activation (e.g., lymphocyte activation,B cell activation) or unwanted cytokine production in a subjectfollowing repeated administration of a messenger RNA (mRNA) to thesubject, the method comprising:

(i) administering to the subject intravenously a first dose of a mRNA,e.g., chemically modified mRNA (mmRNA) encapsulated in an LNP, whereinthe mRNA, e.g., mmRNA comprises at least one miR-126 and/or miR-142microRNA binding site, and wherein the mRNA, e.g., mmRNA comprises oneor more modified nucleobases;

(ii) detecting a level of immune cell activation in a sample from thesubject; and

(iii) administering to the subject intravenously a second dose of themRNA, e.g., mmRNA encapsulated in an LNP when the level of immune cellactivation in the sample is diminished, such that unwanted immune cellactivation or unwanted cytokine production is reduced or inhibited inthe subject.

In certain embodiments, the mRNA, e.g., mmRNA, encodes a polypeptide ofinterest (e.g., a therapeutic agent), wherein unwanted immune cellactivation or unwanted cytokine production occurs in response to thepolypeptide of interest.

ADA Assays

ADA assays (bioassays) can be used to assay for both neutralizingantibodies (NABs) and non-neutralizing, binding antibodies (BABs). NABassays can include both cell based assays, for example, cellproliferation assays, biomarker assays, gene expression assays, genereporter assays, antibody-dependent cell-mediated cytotoxicity (ADCC)assays, complement-dependent cytotoxicity (CDC) assays, and the like, aswell as non-cell based assays, for example, competitive ligand-binding(CLBA) assays, surface plasmon resonance (SPR), enzyme-linkedimmunosorbent assay (ELISA), electro-chemiluminescence (ECL), e.g.,electro-chemiluminescence immunoassay (ECLIA), dissociation-enhancedlanthanide fluorescent immunoassay (DELFIA®), Gyros® anti-drug antibody(ADA) immunoassays, fluorescent-enzyme immunoassay (FEIA),ristocetin-induced platelet aggregation (RIPA), and the like.

In exemplary aspects, the therapeutic regimen can include conducting oneor more ADA assays before or during a therapeutic regimen. In exemplaryembodiments, the ADA assay is a NAB assay. In such instances, thebioassay should be related to product mechanism of action, otherwise theassay will not be informative as to the effect of NAB on clinicalpharmacology. In preferred embodiments, cell-based NABs are featured inthe therapeutic regimen of the disclosure. If neutralizing cell-basedassays are not feasible/available competitive ligand binding assays oralternatives may be suitable. However, when these are used, it ispreferably demonstrated that the assays reflect neutralizingcapacity/potential in an appropriate manner.

In addition to directly measuring the ADA response, the level of immunecell activation also can be evaluated as a measure of a developingantibody response. The level of immune cell activation can be evalutedby essentially any method established in the art for assessing immunecell activation, such as the frequency of an activated immune cellpopulation, typically assessed by detection of cells expressingcell-surface activation markers, or levels of production of one or morecytokines indicative of immune cell activation. In one embodiment, theimmune cell activation is B cell activation, wherein the level of B cellactivation is determined by measuring the frequency of activated Bcells, such as the frequency of activated B cells among the splenic Bcell population. B cell surface markers indicative of B cell activationare well known in the art (see e.g., Maddalay, R. et al. (2010) FEBSLetters 584:4883-4894). In one embodiment, B cell activation isdetermined by frequency of CD19⁺ CD86⁺ CD69⁺ B cells. In anotherembodiment, the immune cell activation is B cell activation, wherein thelevel of B cell activation is determined by cytokine secretion, such asby secretion of interleukin-6 (IL-6), tumor necrosis factor α (TNF-α) orinterferon-γ (IFN-γ), e.g., in the serum of treated subjects. In oneembodiment, B cell activation is determined by secretion of IL-6, e.g.,in the serum of treated subjects. In other embodiments, the unwantedcytokine production that is reduced or inhibited is production ofinterleukin-6 (IL-6), tumor necrosis factor α (TNF-α) or interferon-γ(IFN-γ), e.g., in the serum of treated subjects. In another embodiment,the unwanted cytokine production that is reduced or inhibited isproduction of interleukin-6 (IL-6).

Administration of Pharmaceutical Compositions

A pharmaceutical composition including one or more RNAs, e.g., mRNAs, ofthe disclosure may be administered to a subject by any suitable route.In some embodiments, compositions of the disclosure are administered byone or more of a variety of routes, including parenteral (e.g.,subcutaneous, intracutaneous, intravenous, intraperitoneal,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional, or intracranial injection, aswell as any suitable infusion technique), oral, trans- or intra-dermal,interdermal, rectal, intravaginal, topical (e.g. by powders, ointments,creams, gels, lotions, and/or drops), mucosal, nasal, buccal, enteral,vitreal, intratumoral, sublingual, intranasal; by intratrachealinstillation, bronchial instillation, and/or inhalation; as an oralspray and/or powder, nasal spray, and/or aerosol, and/or through aportal vein catheter. In some embodiments, a composition may beadministered intravenously, intramuscularly, intradermally,intra-arterially, intratumorally, subcutaneously, or by inhalation.However, the present disclosure encompasses the delivery of compositionsof the disclosure by any appropriate route taking into considerationlikely advances in the sciences of drug delivery. In general, the mostappropriate route of administration will depend upon a variety offactors including the nature of the pharmaceutical composition includingone or more mRNAs (e.g., its stability in various bodily environmentssuch as the bloodstream and gastrointestinal tract), and the conditionof the patient (e.g., whether the patient is able to tolerate particularroutes of administration). In one embodiment, the composition isadministered parenterally. In another embodiment, the composition isadministered intravenously. In another embodiment, the composition isadministered intratumorally.

In certain embodiments, compositions of the disclosure may beadministered at dosage levels sufficient to deliver from about 0.0001mg/kg to about 10 mg/kg, from about 0.001 mg/kg to about 10 mg/kg, fromabout 0.005 mg/kg to about 10 mg/kg, from about 0.01 mg/kg to about 10mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about 1 mg/kg toabout 10 mg/kg, from about 2 mg/kg to about 10 mg/kg, from about 5 mg/kgto about 10 mg/kg, from about 0.0001 mg/kg to about 5 mg/kg, from about0.001 mg/kg to about 5 mg/kg, from about 0.005 mg/kg to about 5 mg/kg,from about 0.01 mg/kg to about 5 mg/kg, from about 0.1 mg/kg to about 10mg/kg, from about 1 mg/kg to about 5 mg/kg, from about 2 mg/kg to about5 mg/kg, from about 0.0001 mg/kg to about 1 mg/kg, from about 0.001mg/kg to about 1 mg/kg, from about 0.005 mg/kg to about 1 mg/kg, fromabout 0.01 mg/kg to about 1 mg/kg, or from about 0.1 mg/kg to about 1mg/kg in a given dose, where a dose of 1 mg/kg provides 1 mg of mRNA ornanoparticle per 1 kg of subject body weight. In particular embodiments,a dose of about 0.005 mg/kg to about 5 mg/kg of mRNA or nanoparticle ofthe disclosure may be administrated. In particular embodiments, a doseof about 0.002 mg/kg to about 2 mg/kg of mRNA or nanoparticle of thedisclosure may be administrated. In particular embodiments, a dose ofabout 0.02 mg/kg to about 0.2 mg/kg of mRNA or nanoparticle of thedisclosure may be administrated.

A dose may be administered one or more times per day, in the same or adifferent amount, to obtain a desired level of mRNA, expression and/oreffect (e.g., a therapeutic effect). The desired dosage may bedelivered, for example, three times a day, two times a day, once a day,every other day, every third day, every week, every two weeks, everythree weeks, or every four weeks. In certain embodiments, the desireddosage may be delivered using multiple administrations (e.g., two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, or more administrations). In some embodiments, asingle dose may be administered, for example, prior to or after asurgical procedure or in the instance of an acute disease, disorder, orcondition.

The specific therapeutically effective, prophylactically effective, orotherwise appropriate dose level for any particular patient will dependupon a variety of factors including the severity and identify of adisorder being treated, if any; the one or more mRNAs employed; thespecific composition employed; the age, body weight, general health,sex, and diet of the patient; the time of administration, route ofadministration, and rate of excretion of the specific pharmaceuticalcomposition employed; the duration of the treatment; drugs used incombination or coincidental with the specific pharmaceutical compositionemployed; and like factors well known in the medical arts.

In some embodiments, a pharmaceutical composition of the disclosure maybe administered in combination with another agent, for example, anothertherapeutic agent, a prophylactic agent, and/or a diagnostic agent. By“in combination with,” it is not intended to imply that the agents mustbe administered at the same time and/or formulated for deliverytogether, although these methods of delivery are within the scope of thepresent disclosure. For example, one or more compositions including oneor more different mRNAs may be administered in combination. Compositionscan be administered concurrently with, prior to, or subsequent to, oneor more other desired therapeutics or medical procedures. In general,each agent will be administered at a dose and/or on a time scheduledetermined for that agent. In some embodiments, the present disclosureencompasses the delivery of compositions of the disclosure, or imaging,diagnostic, or prophylactic compositions thereof in combination withagents that improve their bioavailability, reduce and/or modify theirmetabolism, inhibit their excretion, and/or modify their distributionwithin the body.

Exemplary therapeutic agents that may be administered in combinationwith the compositions of the disclosure include, but are not limited to,cytotoxic, chemotherapeutic, and other therapeutic agents. Cytotoxicagents may include, for example, taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, teniposide,vincristine, vinblastine, colchicine, doxorubicin, daunorubicin,dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, puromycin, maytansinoids, rachelmycin, and analogs thereof.Radioactive ions may also be used as therapeutic agents and may include,for example, radioactive iodine, strontium, phosphorous, palladium,cesium, iridium, cobalt, yttrium, samarium, and praseodymium. Othertherapeutic agents may include, for example, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and5-fluorouracil, and decarbazine), alkylating agents (e.g.,mechlorethamine, thiotepa, chlorambucil, rachelmycin, melphalan,carmustine, lomustine, cyclophosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II)(DDP), and cisplatin), anthracyclines (e.g., daunorubicin anddoxorubicin), antibiotics (e.g., dactinomycin, bleomycin, mithramycin,and anthramycin), and anti-mitotic agents (e.g., vincristine,vinblastine, taxol, and maytansinoids).

The particular combination of therapies (therapeutics or procedures) toemploy in a combination regimen will take into account compatibility ofthe desired therapeutics and/or procedures and the desired therapeuticeffect to be achieved. It will also be appreciated that the therapiesemployed may achieve a desired effect for the same disorder (forexample, a composition useful for treating cancer may be administeredconcurrently with a chemotherapeutic agent), or they may achievedifferent effects (e.g., control of any adverse effects).

Other Embodiments

This disclosure relates to the following embodiments:

In some aspects, the disclosure relates to methods of reducing orinhibiting an anti-drug antibody response in a subject, comprisingadministering to the subject a modified messenger RNA (mmRNA) encoding apolypeptide of interest, wherein the mmRNA comprises at least onemiR-142-3p microRNA binding site, and wherein the mmRNA comprises one ormore modified nucleobases, such that an anti-drug antibody response tothe polypeptide of interest is reduced or inhibited in the subject.

In other aspects, the disclosure relates to methods of reducing orinhibiting an anti-drug antibody response following repeatedadministration of a polypeptide of interest to a subject, comprisingadministering to the subject intravenously a first dose of a modifiedmRNA (mmRNA) encoding a polypeptide of interest encapsulated in an LNP,wherein the mmRNA comprises at least one miR-142-3p microRNA bindingsite, and wherein the mmRNA comprises one or more modified nucleobases;and administering to the subject intravenously a second dose of themmRNA encapsulated in an LNP, such that an anti-drug antibody responseto the polypeptide of interest is reduced or inhibited in the subject.

In yet further aspects, the disclosure relates to methods of reducing orinhibiting an anti-drug antibody response following repeatedadministration of a polypeptide of interest to a subject, comprising

(i) administering to the subject intravenously a first dose of amodified mRNA (mmRNA) encoding a polypeptide of interest encapsulated inan LNP, wherein the mmRNA comprises at least one miR-142-3p microRNAbinding site, and wherein the mmRNA comprises one or more modifiednucleobases;

(ii) detecting a level of anti-drug antibodies in a sample from thesubject; and

(iii) administering to the subject intravenously a second dose of themmRNA encapsulated in an LNP when the level of anti-drug antibodies inthe sample is diminished, such that an anti-drug antibody response tothe polypeptide of interest is reduced or inhibited in the subject.

In some aspects, the disclosure relates to methods of reducing orinhibiting drug-related toxicity in a subject, comprising administeringto the subject a modified messenger RNA (mmRNA) encoding a polypeptideof interest, wherein the mmRNA comprises at least one miR-142-3pmicroRNA binding site, and wherein the mmRNA comprises one or moremodified nucleobases, such that drug-related toxicity to the polypeptideof interest is reduced or inhibited in the subject.

In other aspects, the disclosure relates to methods of reducing orinhibiting drug-related toxicity in a subject, comprising administeringto the subject a modified messenger RNA (mmRNA) encoding a polypeptideof interest, wherein the mmRNA comprises at least one miR-126 microRNAbinding site, and wherein the mmRNA comprises one or more modifiednucleobases, such that drug-related toxicity to the polypeptide ofinterest is reduced or inhibited in the subject.

In some embodiments of the foregoing aspects, drug-related toxicity tothe polypeptide of interest is decreased blood cell counts (cytopenia)in the subject. In other embodiments of the foregoing aspects,drug-related toxicity to the polypeptide of interest is autoimmunity inthe subject. In further embodiments of the foregoing aspects,drug-related toxicity to the polypeptide of interest is complementmediated effects in the subject. In some embodiments of the foregoingaspects, drug-related toxicity to the polypeptide of interest isdecreased hematopoiesis in the subject. In other embodiments of theforegoing aspects, drug-related toxicity is renal toxicity or livertoxicity.

In some aspects, the disclosure relates to methods of reducing orinhibiting an anti-drug antibody response in a subject, comprisingadministering to the subject a modified messenger RNA (mmRNA) encoding apolypeptide of interest, wherein the mmRNA comprises at least onemiR-126 microRNA binding site, and wherein the mmRNA comprises one ormore modified nucleobases, such that an anti-drug antibody response tothe polypeptide of interest is reduced or inhibited in the subject.

In other aspects, the disclosure relates to methods of reducing orinhibiting an anti-drug antibody response following repeatedadministration of a polypeptide of interest to a subject, comprisingadministering to the subject intravenously a first dose of a modifiedmRNA (mmRNA) encoding a polypeptide of interest encapsulated in an LNP,wherein the mmRNA comprises at least one miR-126 microRNA binding site,and wherein the mmRNA comprises one or more modified nucleobases; andadministering to the subject intravenously a second dose of the mmRNAencapsulated in an LNP, such that an anti-drug antibody response to thepolypeptide of interest is reduced or inhibited in the subject.

In yet further aspects, the disclosure relates to methods of reducing orinhibiting an anti-drug antibody response following repeatedadministration of a polypeptide of interest to a subject, comprising

(i) administering to the subject intravenously a first dose of amodified mRNA (mmRNA) encoding a polypeptide of interest encapsulated inan LNP, wherein the mmRNA comprises at least one miR-126 microRNAbinding site, and wherein the mmRNA comprises one or more modifiednucleobases;

(ii) detecting a level of anti-drug antibodies in a sample from thesubject; and

(iii) administering to the subject intravenously a second dose of themmRNA encapsulated in an LNP when the level of anti-drug antibodies inthe sample is diminished, such that an anti-drug antibody response tothe polypeptide of interest is reduced or inhibited in the subject.

In some aspects, the disclosure relates to methods of reducing orinhibiting unwanted immune cell activation in a subject administered amessenger RNA (mRNA) encoding a polypeptide of interest, the methodcomprising administering to the subject a chemically modified mRNAencoding the polypeptide of interest, wherein the chemically modifiedmRNA comprises at least one microRNA binding site for a microRNAexpressed in immune cells, and wherein the chemically modified mRNAcomprises one or more modified nucleobases, such that unwanted immunecell activation is reduced or inhibited in the subject.

In some embodiments of the foregoing aspects, reduction or inhibition ofunwanted immune cell activation is determined compared to controladministration of a chemically modified mRNA lacking the at least onemicroRNA binding site. In other embodiments of the foregoing aspects,the reduction or inhibition of unwanted immune cell activation isreduction or inhibition of lymphocyte activation.

In some embodiments of the foregoing aspects, the reduction orinhibition of lymphocyte activation is reduction or inhibition of B cellactivation. In other embodiments of the foregoing aspects, reduction orinhibition of B cell activation is determined by frequency of CD19⁺CD86⁺ CD69⁺ B cells.

In some embodiments of the foregoing aspects, the reduction orinhibition of unwanted immune cell activation causes reduced orinhibited cytokine production. In some embodiments of the foregoingaspects, immune cell activation is decreased by at least 10%. In furtherembodiments of the foregoing aspects, immune cell activation isdecreased by at least 25%. In some embodiments of the foregoing aspects,immune cell activation is decreased by at least 50%. In otherembodiments of the foregoing aspects, wherein immune cell activation isdecreased without a corresponding decrease in expression of thepolypeptide of interest encoded by the chemically modified mRNA.

In some aspects, the disclosure relates to methods of reducing orinhibiting unwanted immune cell activation in a subject administered amessenger RNA (mRNA) encoding a polypeptide of interest, comprisingadministering to the subject intravenously a first dose of a chemicallymodified mRNA encapsulated in an lipid nanoparticle (LNP), wherein thechemically modified mRNA comprises at least one microRNA binding sitefor a microRNA expressed in immune cells, and wherein the chemicallymodified mRNA comprises one or more modified nucleobases; andadministering to the subject intravenously a second dose of thechemically modified mRNA encapsulated in an LNP, such that unwantedimmune cell activation is reduced or inhibited in the subject.

In other aspects, the disclosure relates to methods of reducing orinhibiting unwanted immune cell activation in a subject followingrepeated administration of a messenger RNA (mRNA) encoding a polypeptideof interest to the subject, comprising

(i) administering to the subject intravenously a first dose of achemically modified mRNA encapsulated in a lipid nanoparticle (LNP),wherein the chemically modified mRNA comprises at least one microRNAbinding site for a microRNA expressed in immune cells,

and wherein the chemically modified mRNA comprises one or more modifiednucleobases;

(ii) detecting a level of immune cell activation in a sample from thesubject; and

(iii) administering to the subject intravenously a second dose of thechemically modified mRNA encapsulated in an LNP when the level of immunecell activation in the sample is diminished, such that unwanted immunecell activation is reduced or inhibited in the subject.

In some embodiments of the foregoing aspects, the reduced or inhibitedunwanted immune cell activation is reduced or inhibited B cellactivation. In some embodiments of the foregoing aspects, the reduced orinhibited unwanted immune cell activation causes reduced or inhibitedcytokine production.

In some aspects, the disclosure relates to methods of reducing orinhibiting unwanted cytokine production in a subject administered amessenger RNA (mRNA) encoding a polypeptide of interest, the methodcomprising administering to the subject a chemically modified mRNAencoding the polypeptide of interest, wherein the chemically modifiedmRNA comprises at least one microRNA binding site for a microRNAexpressed in immune cells, and wherein the chemically modified mRNAcomprises one or more modified nucleobases, such that unwanted cytokineproduction is reduced or inhibited in the subject.

In some embodiments of the foregoing aspects, reduction or inhibition ofunwanted cytokine production is determined compared to controladministration of a chemically modified mRNA lacking the at least onemicroRNA binding site for a microRNA expressed in immune cells. In otherembodiments of the foregoing aspects, the reduced or inhibited cytokineproduction is reduced or inhibited production of interleukin-6 tumornecrosis factor □ (TNF-□) or interferon-□ (IFN-□). In some embodimentsof the foregoing aspects, the reduced or inhibited cytokine productionis reduced or inhibited production of interleukin-6 (IL-6).

In some embodiments of the foregoing aspects, cytokine production isdecreased by at least 10%. In some embodiments of the foregoing aspects,cytokine production is decreased by at least 25%. In some embodiments ofthe foregoing aspects, cytokine production is decreased by at least 50%.In some embodiments of the foregoing aspects, cytokine production isdecreased without a corresponding decrease in expression of thepolypeptide of interest encoded by the chemically modified mRNA.

In yet further aspects, the disclosure relates to methods of reducing orinhibiting accelerated blood clearance in a subject repeatedlyadministered a messenger RNA (mRNA) encoding a polypeptide of interestencapsulated in an lipid nanoparticle (LNP), the method comprisingadministering to the subject a chemically modified mRNA encoding thepolypeptide of interest encapsulated in an lipid nanoparticle (LNP),wherein the chemically modified mRNA comprises at least one microRNAbinding site for a microRNA expressed in immune cells, and wherein thechemically modified mRNA comprises one or more modified nucleobases,such that accelerated blood clearance is reduced or inhibited in thesubject upon repeat administration.

In some aspects, the disclosure relates to methods of reducing orinhibiting accelerated blood clearance in a subject administered amessenger RNA (mRNA) encoding a polypeptide of interest encapsulated inan lipid nanoparticle (LNP), comprising administering to the subjectintravenously a first dose of a chemically modified mRNA encapsulated inan lipid nanoparticle (LNP), wherein the chemically modified mRNAcomprises at least one microRNA binding site for a microRNA expressed inimmune cells, and wherein the chemically modified mRNA comprises one ormore modified nucleobases; and administering to the subjectintravenously a second dose of the chemically modified mRNA encapsulatedin an LNP, such that accelerated blood clearance is reduced or inhibitedin the subject.

In other aspects, the disclosure relates to methods of reducing orinhibiting accelerated blood clearance in a subject administeredmultiple doses of a messenger RNA (mRNA) encoding a polypeptide ofinterest encapsulated in an lipid nanoparticle (LNP), the methodcomprising administering to the subject a chemically modified mRNAencoding the polypeptide of interest encapsulated in an lipidnanoparticle (LNP), wherein the chemically modified mRNA comprises atleast one microRNA binding site for a microRNA expressed in immunecells, and wherein the chemically modified mRNA comprises one or moremodified nucleobases, such that accelerated blood clearance is reducedor inhibited in the subject upon administration of one or moresubsequent doses.

In yet further aspects, the disclosure relates to methods of reducing orinhibiting accelerated blood clearance in a subject administered amessenger RNA (mRNA) encoding a polypeptide of interest encapsulated inan lipid nanoparticle (LNP), the method comprising administering to thesubject a chemically modified mRNA encoding the polypeptide of interestencapsulated in an lipid nanoparticle (LNP), wherein the chemicallymodified mRNA comprises at least one microRNA binding site for amicroRNA expressed in immune cells, and wherein the chemically modifiedmRNA comprises one or more modified nucleobases, such that acceleratedblood clearance is reduced or inhibited in the subject uponadministration of a subsequent dose of the mRNA.

In some aspects, the disclosure relates to methods of reducing orinhibiting accelerated blood clearance in a subject repeatedlyadministered a messenger RNA (mRNA) encoding a polypeptide of interestencapsulated in an lipid nanoparticle (LNP), the method comprisingadministering to the subject a chemically modified mRNA encoding thepolypeptide of interest encapsulated in an lipid nanoparticle (LNP),wherein the chemically modified mRNA comprises at least one microRNAbinding site for a microRNA expressed in immune cells, wherein thechemically modified mRNA comprises one or more modified nucleobases, andwherein the LNP does not activate B cells and/or does not induceproduction of IgM molecules capable of binding to the LNP, such thataccelerated blood clearance is reduced or inhibited in the subject uponrepeat administration.

In other aspects, the disclosure relates to methods of reducing orinhibiting accelerated blood clearance in a subject administered amessenger RNA (mRNA) encoding a polypeptide of interest encapsulated inan lipid nanoparticle (LNP), comprising administering to the subjectintravenously a first dose of a chemically modified mRNA encapsulated inan lipid nanoparticle (LNP), wherein the chemically modified mRNAcomprises at least one microRNA binding site for a microRNA expressed inimmune cells, and wherein the chemically modified mRNA comprises one ormore modified nucleobases; and administering to the subjectintravenously a second dose of the chemically modified mRNA encapsulatedin an LNP, wherein the LNP does not activate B cells and/or does notinduce production of IgM molecules capable of binding to the LNP, suchthat accelerated blood clearance is reduced or inhibited in the subject.

In yet further aspects, the disclosure relates to methods of reducing orinhibiting accelerated blood clearance in a subject administeredmultiple doses of a messenger RNA (mRNA) encoding a polypeptide ofinterest encapsulated in an lipid nanoparticle (LNP), the methodcomprising administering to the subject a chemically modified mRNAencoding the polypeptide of interest encapsulated in an lipidnanoparticle (LNP), wherein the chemically modified mRNA comprises atleast one microRNA binding site for a microRNA expressed in immunecells, wherein the chemically modified mRNA comprises one or moremodified nucleobases, and wherein the LNP does not activate B cellsand/or does not induce production of IgM molecules capable of binding tothe LNP, such that accelerated blood clearance is reduced or inhibitedin the subject upon administration of one or more subsequent doses.

In some aspects, the disclosure relates to methods of reducing orinhibiting accelerated blood clearance in a subject administered amessenger RNA (mRNA) encoding a polypeptide of interest encapsulated inan lipid nanoparticle (LNP), the method comprising administering to thesubject a chemically modified mRNA encoding the polypeptide of interestencapsulated in an lipid nanoparticle (LNP), wherein the chemicallymodified mRNA comprises at least one microRNA binding site for amicroRNA expressed in immune cells, wherein the chemically modified mRNAcomprises one or more modified nucleobases, and wherein the LNP does notactivate B cells and/or does not induce production of IgM moleculescapable of binding to the LNP, such that accelerated blood clearance isreduced or inhibited in the subject upon administration of a subsequentdose of the mRNA.

In other aspects, the disclosure relates to methods of reducing orinhibiting production of IgM molecules that recognize polyethyleneglycol (PEG) in a subject repeatedly administered a messenger RNA (mRNA)encoding a polypeptide of interest encapsulated in an lipid nanoparticle(LNP), the method comprising administering to the subject a chemicallymodified mRNA encoding the polypeptide of interest encapsulated in anlipid nanoparticle (LNP), wherein the chemically modified mRNA comprisesat least one microRNA binding site for a microRNA expressed in immunecells, and wherein the chemically modified mRNA comprises one or moremodified nucleobases, such that production of IgM molecules thatrecognize PEG are reduced or inhibited in the subject upon repeatadministration.

In some aspects, the disclosure relates to methods of reducing orinhibiting production of IgM molecules that recognize polyethyleneglycol (PEG) in a subject administered a messenger RNA (mRNA) encoding apolypeptide of interest encapsulated in an lipid nanoparticle (LNP),comprising administering to the subject intravenously a first dose of achemically modified mRNA encapsulated in an lipid nanoparticle (LNP),wherein the chemically modified mRNA comprises at least one microRNAbinding site for a microRNA expressed in immune cells, and wherein thechemically modified mRNA comprises one or more modified nucleobases; andadministering to the subject intravenously a second dose of thechemically modified mRNA encapsulated in an LNP, such that production ofIgM molecules that recognize PEG are reduced or inhibited in thesubject.

In other aspects, the disclosure relates to methods of reducing orinhibiting production of IgM molecules that recognize polyethyleneglycol (PEG) in a subject administered multiple doses of a messenger RNA(mRNA) encoding a polypeptide of interest encapsulated in an lipidnanoparticle (LNP), the method comprising administering to the subject achemically modified mRNA encoding the polypeptide of interestencapsulated in an lipid nanoparticle (LNP), wherein the chemicallymodified mRNA comprises at least one microRNA binding site for amicroRNA expressed in immune cells, and wherein the chemically modifiedmRNA comprises one or more modified nucleobases, such that production ofIgM molecules that recognize PEG are reduced or inhibited in the subjectupon administration of one or more subsequent doses.

In some aspects, the disclosure relates to methods of reducing orinhibiting production of IgM molecules that recognize polyethyleneglycol (PEG) in a subject administered a messenger RNA (mRNA) encoding apolypeptide of interest encapsulated in an lipid nanoparticle (LNP), themethod comprising administering to the subject a chemically modifiedmRNA encoding the polypeptide of interest encapsulated in an lipidnanoparticle (LNP), wherein the chemically modified mRNA comprises atleast one microRNA binding site for a microRNA expressed in immunecells, and wherein the chemically modified mRNA comprises one or moremodified nucleobases, such that production of IgM molecules thatrecognize PEG are reduced or inhibited in the subject uponadministration of a subsequent dose of the mRNA.

In yet further aspects, the disclosure relates to methods of reducing orinhibiting activation of B1a cells in a subject repeatedly administereda messenger RNA (mRNA) encoding a polypeptide of interest encapsulatedin an lipid nanoparticle (LNP), the method comprising administering tothe subject a chemically modified mRNA encoding the polypeptide ofinterest encapsulated in an lipid nanoparticle (LNP), wherein thechemically modified mRNA comprises at least one microRNA binding sitefor a microRNA expressed in immune cells, and wherein the chemicallymodified mRNA comprises one or more modified nucleobases, such thatactivation of B1a cells is reduced or inhibited in the subject uponrepeat administration.

In some aspects, the disclosure relates to methods of reducing orinhibiting activation of B1a cells in a subject administered a messengerRNA (mRNA) encoding a polypeptide of interest encapsulated in an lipidnanoparticle (LNP), comprising administering to the subjectintravenously a first dose of a chemically modified mRNA encapsulated inan lipid nanoparticle (LNP), wherein the chemically modified mRNAcomprises at least one microRNA binding site for a microRNA expressed inimmune cells, and wherein the chemically modified mRNA comprises one ormore modified nucleobases; and administering to the subjectintravenously a second dose of the chemically modified mRNA encapsulatedin an LNP, such that activation of B1a cells is reduced or inhibited inthe subject.

In other aspects, the disclosure relates to methods of reducing orinhibiting activation of B1a cells in a subject administered multipledoses of a messenger RNA (mRNA) encoding a polypeptide of interestencapsulated in an lipid nanoparticle (LNP), the method comprisingadministering to the subject a chemically modified mRNA encoding thepolypeptide of interest encapsulated in an lipid nanoparticle (LNP),wherein the chemically modified mRNA comprises at least one microRNAbinding site for a microRNA expressed in immune cells, and wherein thechemically modified mRNA comprises one or more modified nucleobases,such that activation of B1a cells is reduced or inhibited in the subjectupon administration of one or more subsequent doses.

In some aspects, the disclosure relates to methods of reducing orinhibiting activation of B1a cells in a subject administered a messengerRNA (mRNA) encoding a polypeptide of interest encapsulated in an lipidnanoparticle (LNP), the method comprising administering to the subject achemically modified mRNA encoding the polypeptide of interestencapsulated in an lipid nanoparticle (LNP), wherein the chemicallymodified mRNA comprises at least one microRNA binding site for amicroRNA expressed in immune cells, and wherein the chemically modifiedmRNA comprises one or more modified nucleobases, such that activation ofB1a cells is reduced or inhibited in the subject upon administration ofa subsequent dose of the mRNA.

In other aspects, the disclosure relates to methods of reducing orinhibiting activation of plasmacytoid dendrtic cells in a subjectrepeatedly administered a messenger RNA (mRNA) encoding a polypeptide ofinterest encapsulated in an lipid nanoparticle (LNP), the methodcomprising administering to the subject a chemically modified mRNAencoding the polypeptide of interest encapsulated in an lipidnanoparticle (LNP), wherein the chemically modified mRNA comprises atleast one microRNA binding site for a microRNA expressed in immunecells, and wherein the chemically modified mRNA comprises one or moremodified nucleobases, such that activation of plasmacytoid dendriticcells is reduced or inhibited in the subject upon repeat administration.

In yet further aspects, the disclosure relates to methods of reducing orinhibiting activation of plasmacytoid dendritic cells in a subjectadministered a messenger RNA (mRNA) encoding a polypeptide of interestencapsulated in an lipid nanoparticle (LNP), comprising administering tothe subject intravenously a first dose of a chemically modified mRNAencapsulated in an lipid nanoparticle (LNP), wherein the chemicallymodified mRNA comprises at least one microRNA binding site for amicroRNA expressed in immune cells, and wherein the chemically modifiedmRNA comprises one or more modified nucleobases; and administering tothe subject intravenously a second dose of the chemically modified mRNAencapsulated in an LNP, such that activation of plasmacytoid dendriticcells is reduced or inhibited in the subject.

In some aspects, the disclosure relates to methods of reducing orinhibiting activation of plasmacytoid dendritic cells in a subjectadministered multiple doses of a messenger RNA (mRNA) encoding apolypeptide of interest encapsulated in an lipid nanoparticle (LNP), themethod comprising administering to the subject a chemically modifiedmRNA encoding the polypeptide of interest encapsulated in an lipidnanoparticle (LNP), wherein the chemically modified mRNA comprises atleast one microRNA binding site for a microRNA expressed in immunecells, and wherein the chemically modified mRNA comprises one or moremodified nucleobases, such that activation of plasmacytoid dendriticcells is reduced or inhibited in the subject upon administration of oneor more subsequent doses.

In other aspects, the disclosure relates to methods of reducing orinhibiting activation of plasmacytoid dendritic cells in a subjectadministered a messenger RNA (mRNA) encoding a polypeptide of interestencapsulated in an lipid nanoparticle (LNP), the method comprisingadministering to the subject a chemically modified mRNA encoding thepolypeptide of interest encapsulated in an lipid nanoparticle (LNP),wherein the chemically modified mRNA comprises at least one microRNAbinding site for a microRNA expressed in immune cells, and wherein thechemically modified mRNA comprises one or more modified nucleobases,such that activation of plasmacytoid dendritic cells is reduced orinhibited in the subject upon administration of a subsequent dose of themRNA.

In some embodiments of the foregoing aspects, the mRNA encoding apolypeptide of interest encapsulated in a lipid nanoparticle (LNP) doesnot activate B cells and/or does not induce production of IgM moleculescapable of binding to the LNP. In some embodiments of the foregoingaspects, the mRNA encoding a polypeptide of interest encapsulated in alipid nanoparticle (LNP) does not activate B cells. In some embodimentsof the foregoing aspects, the mRNA encoding a polypeptide of interestencapsulated in a lipid nanoparticle (LNP) does not induce production ofIgM molecules capable of binding to the LNP.

In some embodiments of the foregoing aspects, reduction or inhibition ofaccelerated blood clearance is determined compared to controladministration of a chemically modified mRNA lacking the at least onemicroRNA binding site encapsulated in a lipid nanoparticle (LNP). Insome embodiments of the foregoing aspects, accelerated blood clearanceis reduced or inhibited without a corresponding reduction or inhibitionin expression of the polypeptide of interest encoded by the chemicallymodified mRNA. In some embodiments of the foregoing aspects, theinterval between two consecutive doses is less than 2 weeks. In someembodiments of the foregoing aspects, the interval between twoconsecutive doses is less than 1 week.

In some embodiments of the foregoing aspects, the IgM moleculesrecognize polyethylene glycol (PEG).

In any of the foregoing aspects, the mmRNA described herein isadministered intravenously encapsulated in a lipid nanoparticle. In anyof the foregoing aspects, the mmRNA described herein is administered byonce weekly infusion.

In any of the foregoing aspects, the mmRNA described herein comprises a5′ UTR, a codon optimized open reading frame encoding the polypeptide ofinterest, a 3′ UTR comprising the at least one miR-142-3p microRNAbinding site, and a 3′ tailing region of linked nucleosides. In someembodiments, the mmRNA described herein comprises a 5′ UTR and 3′UTRwhich are heterologous to the coding region. In some embodiments, themmRNA described herein is fully modified. In some embodiments, the mmRNAdescribed herein is fully modified for a particular chemicalmodification.

In any of the foregoing aspects, the mmRNA described herein comprisespseudouridine (ψ), pseudouridine (ψ) and 5-methyl-cytidine (m⁵C),1-methyl-pseudouridine (m¹ψ), 1-methyl-pseudouridine (m¹ψ) and5-methyl-cytidine (m⁵C), 2-thiouridine (s²U), 2-thiouridine and5-methyl-cytidine (m⁵C), 5-methoxy-uridine (mo⁵U), 5-methoxy-uridine(mo⁵U) and 5-methyl-cytidine (m⁵C), 2′-O-methyl uridine, 2′-O-methyluridine and 5-methyl-cytidine (m⁵C), N6-methyl-adenosine (m⁶A) orN6-methyl-adenosine (m⁶A) and 5-methyl-cytidine (m⁵C).

In any of the foregoing aspects, the mmRNA described herein comprisespseudouridine (ψ), N1-methylpseudouridine (m¹ψ), 2-thiouridine,4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methoxyuridine, or 2′-O-methyl uridine, or combinations thereof.

In some embodiments, the mmRNA described herein comprises1-methyl-pseudouridine (m¹ψ), 5-methoxy-uridine (mo⁵U),5-methyl-cytidine (m⁵C), pseudouridine (w), α-thio-guanosine, orα-thio-adenosine, or combinations thereof.

In any of the foregoing aspects, the polypeptide of interest is atherapeutic protein, cytokine, growth factor, antibody or fusionprotein.

In any of the foregoing aspects, the lipid nanoparticle is a liposome.In some embodiments, the lipid nanoparticle comprises a cationic and/orionizable lipid. In some embodiments, the cationic and/or ionizablelipid is DLin-KC2-DMA or DLin-MC3-DMA.

In any of the foregoing aspects, mmRNA comprises at least two microRNAbinding sites, wherein at least one of the microRNA binding sites is amiR-142-3p microRNA binding site. In any of the foregoing aspects, themmRNA comprises a miR-142-3p binding site and a second microRNA bindingsite for a miR selected from the group consisting of miR-142-5p,miR-146-3p, miR-146-5p, miR-155, miR-126, miR-16, miR-21, miR-223,miR-24 and miR-27.

In any of the foregoing aspects, the chemically modified mRNA comprisesa 5′ UTR, a codon optimized open reading frame encoding the polypeptideof interest, a 3′ UTR comprising the at least one microRNA binding site,and a 3′ tailing region of linked nucleosides.

In any of the foregoing aspects, the codon optimized open reading frameencoding the polypeptide of interest comprises a stop codon and whereinthe at least one microRNA binding site is located within the 3′ UTR1-100 nucleotides after the stop codon. In some embodiments, the codonoptimized open reading frame encoding the polypeptide of interestcomprises a stop codon and wherein the at least one microRNA bindingsite is located within the 3′ UTR at least 50 nucleotides after the stopcodon.

In any of the foregoing aspects, the chemically modified mRNA comprisesa 5′ UTR, a codon optimized open reading frame encoding the polypeptideof interest, a 3′ UTR comprising the at least one microRNA binding sitefor a microRNA expressed in immune cells, and a 3′ tailing region oflinked nucleosides.

In any of the foregoing aspects, the codon optimized open reading frameencoding the polypeptide of interest comprises a stop codon and whereinthe at least one miR-142-3p microRNA binding site is located within the3′ UTR 30-50 nucleotides after the stop codon. In some embodiments, thecodon optimized open reading frame encoding the polypeptide of interestcomprises a stop codon and wherein the at least one miR-142-3p microRNAbinding site is located within the 3′ UTR at least 50 nucleotides afterthe stop codon.

In any of the foregoing aspects, the codon optimized open reading frameencoding the polypeptide of interest comprises a stop codon and whereinthe at least one miR-126 microRNA binding site is located within the 3′UTR 30-50 nucleotides after the stop codon. In some embodiments, thecodon optimized open reading frame encoding the polypeptide of interestcomprises a stop codon and wherein the at least one miR-126 microRNAbinding site is located within the 3′ UTR at least 50 nucleotides afterthe stop codon.

In any of the foregoing aspects, the mmRNA comprises a 5′ UTR, a codonoptimized open reading frame encoding the polypeptide of interest, a 3′UTR comprising the at least one miR-126 microRNA binding site, and a 3′tailing region of linked nucleosides. In any of the foregoing aspects,the mmRNA comprises at least two microRNA binding sites. In someembodiments, the mmRNA comprises at least two microRNA binding sites,wherein at least one of the microRNA binding sites is a miR-126 microRNAbinding site. In some embodiments, the mmRNA comprises a miR-126 bindingsite and a second microRNA binding site for a miR selected from thegroup consisting of miR-142-3p, miR-142-5p, miR-146-3p, miR-146-5p,miR-155, miR-16, miR-21, miR-223, miR-24 and miR-27. In someembodiments, the chemically modified mRNA comprises a miR-126 bindingsite and a miR-142 binding site. In some embodiments, the mmRNAconstruct comprises a miR-126 binding site and a miR-142-3p bindingsite.

In any of the foregoing aspects, the microRNA binding site is amiR-142-3p binding site. In some embodiments, the miR-142-3p bindingsite comprises the sequence shown in SEQ ID NO: 3.

In any of the foregoing aspects, the microRNA binding site is a miR-126microRNA binding site. In some embodiments, the miR-126 binding sitecomprises the sequence shown in SEQ ID NO: 26.

In any of the foregoing aspects, the microRNA binding site is a miR-155microRNA binding site. In some embodiments, the miR-155 binding sitecomprises the sequence shown in SEQ ID NO: 35.

In any of the foregoing aspects, the microRNA binding site binds amicroRNA expressed in myeloid cells. In any of the foregoing aspects,the microRNA binding site binds a microRNA expressed in plasmacytoiddendritic cells. In any of the foregoing aspects, the microRNA bindingsite binds a microRNA expressed in macrophages.

Definitions

Accelerated blood clearance (ABC): As used herein, “accelerated bloodclearance” or “ABC” refers to a phenomenon in which certain exogenouslyadministered agents are rapidly cleared from the blood upon second andsubsequence administrations.

Administering: As used herein, “administering” refers to a method ofdelivering a composition to a subject or patient. A method ofadministration may be selected to target delivery (e.g., to specificallydeliver) to a specific region or system of a body. For example, anadministration may be parenteral (e.g., subcutaneous, intracutaneous,intravenous, intraperitoneal, intramuscular, intraarticular,intraarterial, intrasynovial, intrasternal, intrathecal, intralesional,or intracranial injection, as well as any suitable infusion technique),oral, trans- or intra-dermal, interdermal, rectal, intravaginal, topical(e.g. by powders, ointments, creams, gels, lotions, and/or drops),mucosal, nasal, buccal, enteral, vitreal, intratumoral, sublingual,intranasal; by intratracheal instillation, bronchial instillation,and/or inhalation; as an oral spray and/or powder, nasal spray, and/oraerosol, and/or through a portal vein catheter.

Anti-drug antibody: As used herein, the term “anti-drug antibody”, or“ADA”, refers to antibodies generated in a subject against a therapeuticprotein present in the subject. A classical anti-drug antibody (ADA)response is understood in the art to result from systemic administrationof a recombinant therapeutic protein to the subject. Moreover, as usedherein with respect to mRNA therapeutics, an ADA response is intended toencompass the antibody responses observed in the herein-described animalstudies wherein antibodies were generated that bind to the therapeuticprotein encoded by the mRNA therapeutic (i.e., antibodies generatedagainst the protein encoded by the mRNA drug). Such antibody responsesto the therapeutic protein encoded by the mRNA drug are also referred toas anti-protein antibody (APA) responses, which terminology can be usedinterchangeably herein with ADA responses.

Apoptosis: As used herein, “apoptosis” refers to a form of cell death inwhich a programmed sequence of events leads to the death of a cell.Hallmarks of apoptosis include morphological changes, cell shrinkage,caspase activation, nuclear and cytoplasmic condensation, andalterations in plasma membrane topology. Biochemically, apoptotic cellsare characterized by increased intracellular calcium concentration,fragmentation of chromosomal DNA, and expression of novel cell surfacecomponents. In particular embodiments, a cell undergoing apoptosis mayundergo mitochondrial outer membrane permeabilization (MOMP).

Approximately, about: As used herein, the terms “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Binding Antibody (BAB): As used herein, a “binding antibody” or “BAB”refers to an antibody that is capable of binding to, i.e., interactingwith, a target antigen, such as a therapeutic protein. The term bindingantibody is intended to encompass neutralizing antibodies (i.e.,antibodies that bind to the target antigen and inhibit the functionalactivity of the antigen) and non-neutralizing antibodies (i.e.,antibodies that bind to the target antigen but that do not inhibit thefunctional activity of the antigen).

Cancer: As used herein, “cancer” is a condition involving abnormaland/or unregulated cell growth. The term cancer encompasses benign andmalignant cancers. Exemplary non-limiting cancers include adrenalcortical cancer, advanced cancer, anal cancer, aplastic anemia, bileductcancer, bladder cancer, bone cancer, bone metastasis, brain tumors,brain cancer, breast cancer, childhood cancer, cancer of unknown primaryorigin, Castleman disease, cervical cancer, colorectal cancer,endometrial cancer, esophagus cancer, Ewing family of tumors, eyecancer, gallbladder cancer, gastrointestinal carcinoid tumors,gastrointestinal stromal tumors, gestational trophoblastic disease,Hodgkin disease, Kaposi sarcoma, renal cell carcinoma, laryngeal andhypopharyngeal cancer, acute lymphocytic leukemia, acute myeloidleukemia, chronic lymphocytic leukemia, chronic myeloid leukemia,chronic myelomonocytic leukemia, liver cancer (e.g., hepatocellularcarcinoma), non-small cell lung cancer, small cell lung cancer, lungcarcinoid tumor, lymphoma of the skin, malignant mesothelioma, multiplemyeloma, myelodysplasia syndrome, nasal cavity and paranasal sinuscancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oralcavity and oropharyngeal cancer, osteosarcoma, ovarian cancer,pancreatic cancer, penile cancer, pituitary tumors, prostate cancer,retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma inadult soft tissue, basal and squamous cell skin cancer, melanoma, smallintestine cancer, stomach cancer, testicular cancer, throat cancer,thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvarcancer, Waldenstrom macroglobulinemia, Wilms tumor and secondary cancerscaused by cancer treatment. In particular embodiments, the cancer isliver cancer (e.g., hepatocellular carcinoma) or colorectal cancer. Inother embodiments, the cancer is a blood-based cancer or a hematopoeticcancer.

Conjugated: As used herein, the term “conjugated,” when used withrespect to two or more moieties, means that the moieties are physicallyassociated or connected with one another, either directly or via one ormore additional moieties that serves as a linking agent, to form astructure that is sufficiently stable so that the moieties remainphysically associated under the conditions in which the structure isused, e.g., physiological conditions. In some embodiments, two or moremoieties may be conjugated by direct covalent chemical bonding. In otherembodiments, two or more moieties may be conjugated by ionic bonding orhydrogen bonding.

Contacting: As used herein, the term “contacting” means establishing aphysical connection between two or more entities. For example,contacting a cell with an mRNA or a lipid nanoparticle composition meansthat the cell and mRNA or lipid nanoparticle are made to share aphysical connection. Methods of contacting cells with external entitiesboth in vivo, in vitro, and ex vivo are well known in the biologicalarts. In exemplary embodiments of the disclosure, the step of contactinga mammalian cell with a composition (e.g., an isolated mRNA,nanoparticle, or pharmaceutical composition of the disclosure) isperformed in vivo. For example, contacting a lipid nanoparticlecomposition and a cell (for example, a mammalian cell) that may bedisposed within an organism (e.g., a mammal) may be performed by anysuitable administration route (e.g., parenteral administration to theorganism, including intravenous, intramuscular, intradermal, andsubcutaneous administration). For a cell present in vitro, a composition(e.g., a lipid nanoparticle or an isolated mRNA) and a cell may becontacted, for example, by adding the composition to the culture mediumof the cell and may involve or result in transfection. Moreover, morethan one cell may be contacted by a nanoparticle composition.

Drug-related toxicity: As used herein, the term “drug-related toxicity”,or simply “toxicity”, refers to undesirable in vivo effects that mayresult from expression in a subject of a therapeutic protein encoded byan mRNA, for example as a result of an immune response being stimulatedagainst the encoded therapeutic protein, such as the generation ofantibodies that bind to (and potentially neutralize) the encodedtherapeutic protein. Thus, the term “drug-related toxicity” is intendedto encompass the in vivo adverse effects resulting from an unwantedimmune response against the encoded therapeutic protein, including butnot limited to hematological effects (e.g., hematoxicity), renaleffects, autoimmune effects, liver effects and the like.

Encapsulate: As used herein, the term “encapsulate” means to enclose,surround, or encase. In some embodiments, a compound, polynucleotide(e.g., an mRNA), or other composition may be fully encapsulated,partially encapsulated, or substantially encapsulated. For example, insome embodiments, an mRNA of the disclosure may be encapsulated in alipid nanoparticle, e.g., a liposome.

Effective amount: As used herein, the term “effective amount” of anagent is that amount sufficient to effect beneficial or desired results,for example, clinical results, and, as such, an “effective amount”depends upon the context in which it is being applied. For example, inthe context of administering an agent that treats cancer, an effectiveamount of an agent is, for example, an amount sufficient to achievetreatment, as defined herein, of cancer, as compared to the responseobtained without administration of the agent. In some embodiments, atherapeutically effective amount is an amount of an agent to bedelivered (e.g., nucleic acid, therapeutic agent, diagnostic agentorprophylactic agent) that is sufficient, when administered to a subjectsuffering from or susceptible to an infection, disease, disorder, and/orcondition, to treat, improve symptoms of, diagnose, prevent, and/ordelay the onset of the infection, disease, disorder, and/or condition.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end processing); (3) translation of an RNA into a polypeptide orprotein; and (4) post-translational modification of a polypeptide orprotein.

Identity: As used herein, the term “identity” refers to the overallrelatedness between polymeric molecules, e.g., between polynucleotidemolecules (e.g., DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of the percent identity of twopolynucleotide sequences, for example, can be performed by aligning thetwo sequences for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid sequencesfor optimal alignment and non-identical sequences can be disregarded forcomparison purposes). In certain embodiments, the length of a sequencealigned for comparison purposes is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or 100% of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleotide sequences can be determinedusing the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), whichhas been incorporated into the ALIGN program (version 2.0) using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity between two nucleotide sequences can,alternatively, be determined using the GAP program in the GCG softwarepackage using an NWSgapdna.CMP matrix. Methods commonly employed todetermine percent identity between sequences include, but are notlimited to those disclosed in Carillo, H., and Lipman, D., SIAM JApplied Math., 48:1073 (1988); incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs. Exemplary computer software to determine homologybetween two sequences include, but are not limited to, GCG programpackage, Devereux et al., Nucleic Acids Research, 12(1): 387, 1984,BLASTP, BLASTN, and FASTA, Altschul, S. F. et al., J. Molec. Biol., 215,403, 1990.

Fragment: A “fragment,” as used herein, refers to a portion. Forexample, fragments of proteins may include polypeptides obtained bydigesting full-length protein isolated from cultured cells or obtainedthrough recombinant DNA techniques.

Hematotoxicity: As used herein, the term “hematotoxicity” refers totoxicological events (e.g., resulting from drug-related toxicity)occurring in the hematopoietic system, including but not limited tocytopenias (e.g., reticulocytopenia, thrombocytopenia, neutropenia),decreased hematopoiesis and anemia. While not intended to be limited bymechanism, hematotoxicity in a subject can result from the developmentof an immune response against a therapeutic protein encoded by an mRNAadministered to the subject, e.g., as a result of antibodies beinggenerated against the therapeutic protein, and thus hematotoxicity canbe a subset of drug-related toxicities.

Heterologous: As used herein, “heterologous” indicates that a sequence(e.g., an amino acid sequence or the polynucleotide that encodes anamino acid sequence) is not normally present in a given polypeptide orpolynucleotide. For example, an amino acid sequence that corresponds toa domain or motif of one protein may be heterologous to a secondprotein.

Hydrophobic amino acid: As used herein, a “hydrophobic amino acid” is anamino acid having an uncharged, nonpolar side chain. Examples ofnaturally occurring hydrophobic amino acids are alanine (Ala), valine(Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine(Phe), methionine (Met), and tryptophan (Trp).

Isolated: As used herein, the term “isolated” refers to a substance orentity that has been separated from at least some of the components withwhich it was associated (whether in nature or in an experimentalsetting). Isolated substances may have varying levels of purity inreference to the substances from which they have been associated.Isolated substances and/or entities may be separated from at least about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, or more of the other components with which theywere initially associated. In some embodiments, isolated agents are morethan about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, ormore than about 99% pure. As used herein, a substance is “pure” if it issubstantially free of other components.

Liposome: As used herein, by “liposome” is meant a structure including alipid-containing membrane enclosing an aqueous interior. Liposomes mayhave one or more lipid membranes. Liposomes include single-layeredliposomes (also known in the art as unilamellar liposomes) andmulti-layered liposomes (also known in the art as multilamellarliposomes).

Metastasis: As used herein, the term “metastasis” means the process bywhich cancer spreads from the place at which it first arose as a primarytumor to distant locations in the body. A secondary tumor that arose asa result of this process may be referred to as “a metastasis.” mRNA: Asused herein, an “mRNA” refers to a messenger ribonucleic acid.

An mRNA may be naturally or non-naturally occurring. For example, anmRNA may include modified and/or non-naturally occurring components suchas one or more nucleobases, nucleosides, nucleotides, or linkers. AnmRNA may include a cap structure, a chain terminating nucleoside, a stemloop, a polyA sequence, and/or a polyadenylation signal. An mRNA mayhave a nucleotide sequence encoding a polypeptide. Translation of anmRNA, for example, in vivo translation of an mRNA inside a mammaliancell, may produce a polypeptide. Traditionally, the basic components ofan mRNA molecule include at least a coding region, a 5′-untranslatedregion (5′UTR), a 3′UTR, a 5′ cap and a polyA sequence.

microRNA (miRNA): As used herein, a “microRNA (miRNA)” is a smallnoncoding RNA molecule which may function in post-transcriptionalregulation of gene expression (e.g., by RNA silencing, such as bycleavage of the mRNA, destabilization of the mRNA by shortening itspolyA tail, and/or by interfering with the efficiency of translation ofthe mRNA into a polypeptide by a ribosome). A mature miRNA is typicallyabout 22-23 nucleotides long.

microRNA (miRNA) (miR) binding site: As used herein, a “microRNA (miRNA)(miR) binding site” refers to a miRNA (miR) target site or a miRNA (miR)recognition site, or any nucleotide sequence to which a miRNA (miR)binds or associates. In some embodiments, a miRNA (miR) binding siterepresents a nucleotide location or region of a polynucleotide (e.g., anmRNA) to which at least the “seed” region of a miRNA (miR) binds. Itshould be understood that “binding” may follow traditional Watson-Crickhybridization rules or may reflect any stable association of the miRNAwith the target sequence at or adjacent to the microRNA site. Whenreferring to a miRNA (miR) binding site, a miRNA (miR) sequence is to beunderstood as having complementarity (e.g., partial, substantial, orcomplete (or full) complementarity) with the miRNA that binds to themiRNA binding site. A miRNA (miR) binding site can be partiallycomplementary to a miRNA (miR), e.g., to an endogenous miRNA (miR), asis the case when the miRNA (miR) may exert translational control and/ortranscript stability control of its corresponding mRNA. Alternatively, amiRNA (miR) binding site can be fully complementary (completecomplementarity) to a miRNA (miR), e.g., to an endogenous miRNA (miR),as is the case when the miRNA (miR) may exert post-transcriptionaland/or translational control of its corresponding mRNA. In preferredaspects of the disclosure, a miRNA (miR) binding site is fullycomplementary to a miRNA (miR), e.g., to an endogenous miRNA (miR), andmay cause cleavage of the mRNA comprising said miRNA (miR) in cellsand/or tissues in vivo, where the corresponding miR is expressed, e.g.,endogenously expressed.

miRNA seed: As used herein, a “seed” region of a miRNA refers to asequence in the region of positions 2-8 of a mature miRNA, whichtypically has perfect Watson-Crick complementarity to the miRNA bindingsite. A miRNA seed may include positions 2-8 or 2-7 of a mature miRNA.In some embodiments, a miRNA seed may comprise 7 nucleotides (e.g.,nucleotides 2-8 of a mature miRNA), wherein the seed-complementary sitein the corresponding miRNA binding site is flanked by an adenine (A)opposed to miRNA position 1. In some embodiments, a miRNA seed maycomprise 6 nucleotides (e.g., nucleotides 2-7 of a mature miRNA),wherein the seed-complementary site in the corresponding miRNA bindingsite is flanked by an adenine (A) opposed to miRNA position 1. Whenreferring to a miRNA binding site, a miRNA seed sequence is to beunderstood as having complementarity (e.g., partial, substantial, orcomplete (or full) complementarity) with the seed sequence of the miRNAthat binds to the miRNA binding site.

Modified: As used herein “modified” refers to a changed state orstructure of a molecule of the disclosure. Molecules may be modified inmany ways including chemically, structurally, and functionally. In oneembodiment, the mRNA molecules of the present disclosure are modified bythe introduction of non-natural nucleosides and/or nucleotides, e.g., asit relates to the natural ribonucleotides A, U, G, and C. Noncanonicalnucleotides such as the cap structures are not considered “modified”although they differ from the chemical structure of the A, C, G, Uribonucleotides.

Nanoparticle: As used herein, “nanoparticle” refers to a particle havingany one structural feature on a scale of less than about 1000 nm thatexhibits novel properties as compared to a bulk sample of the samematerial. Routinely, nanoparticles have any one structural feature on ascale of less than about 500 nm, less than about 200 nm, or about 100nm. Also routinely, nanoparticles have any one structural feature on ascale of from about 50 nm to about 500 nm, from about 50 nm to about 200nm or from about 70 to about 120 nm. In exemplary embodiments, ananoparticle is a particle having one or more dimensions of the order ofabout 1-1000 nm. In other exemplary embodiments, a nanoparticle is aparticle having one or more dimensions of the order of about 10-500 nm.In other exemplary embodiments, a nanoparticle is a particle having oneor more dimensions of the order of about 50-200 nm. A sphericalnanoparticle would have a diameter, for example, of between about 50-100or 70-120 nanometers. A nanoparticle most often behaves as a unit interms of its transport and properties. It is noted that novel propertiesthat differentiate nanoparticles from the corresponding bulk materialtypically develop at a size scale of under 1000 nm, or at a size ofabout 100 nm, but nanoparticles can be of a larger size, for example,for particles that are oblong, tubular, and the like. Although the sizeof most molecules would fit into the above outline, individual moleculesare usually not referred to as nanoparticles.

Neutralizing Antibody (NAB): As used herein, a “neutralizing antibody”or “NAB” refers to an antibody that is capable of binding to (i.e.,interacting with) a target antigen, (such as a therapeutic protein) andinhibiting at least one functional activity of the antigen. Binding of aneutralizing antibody to its target antigen may cause partial inhibitionof at least one functional activity of antigen or complete inhibition ofat least one functional activity of the antigen. In certain instances,binding of a neutralizing antibody to its target antigen may causepartial or complete inhibition of all functional activities of thetarget antigen.

Nucleic acid: As used herein, the term “nucleic acid” is used in itsbroadest sense and encompasses any compound and/or substance thatincludes a polymer of nucleotides. These polymers are often referred toas polynucleotides. Exemplary nucleic acids or polynucleotides of thedisclosure include, but are not limited to, ribonucleic acids (RNAs),deoxyribonucleic acids (DNAs), DNA-RNA hybrids, RNAi-inducing agents,RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes,catalytic DNA, RNAs that induce triple helix formation, threose nucleicacids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs),locked nucleic acids (LNAs, including LNA having a β-D-riboconfiguration, α-LNA having an α-L-ribo configuration (a diastereomer ofLNA), 2′-amino-LNA having a 2′-amino functionalization, and2′-amino-α-LNA having a 2′-amino functionalization) or hybrids thereof.

Patient: As used herein, “patient” refers to a subject who may seek orbe in need of treatment, requires treatment, is receiving treatment,will receive treatment, or a subject who is under care by a trainedprofessional for a particular disease or condition. In particularembodiments, a patient is a human patient.

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” isemployed herein to refer to those compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

Pharmaceutically acceptable excipient: The phrase “pharmaceuticallyacceptable excipient,” as used herein, refers any ingredient other thanthe compounds described herein (for example, a vehicle capable ofsuspending or dissolving the active compound) and having the propertiesof being substantially nontoxic and non-inflammatory in a patient.Excipients may include, for example: antiadherents, antioxidants,binders, coatings, compression aids, disintegrants, dyes (colors),emollients, emulsifiers, fillers (diluents), film formers or coatings,flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, and waters of hydration. Exemplary excipients include, butare not limited to: butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide,vitamin A, vitamin E, vitamin C, and xylitol.

Pharmaceutically acceptable salts: As used herein, “pharmaceuticallyacceptable salts” refers to derivatives of the disclosed compoundswherein the parent compound is modified by converting an existing acidor base moiety to its salt form (e.g., by reacting the free base groupwith a suitable organic acid). Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like. Representative acidaddition salts include acetate, acetic acid, adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzene sulfonic acid, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate,hexanoate, hydrobromide, hydrochloride, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. The pharmaceutically acceptable salts of the presentdisclosure include the conventional non-toxic salts of the parentcompound formed, for example, from non-toxic inorganic or organic acids.The pharmaceutically acceptable salts of the present disclosure can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al.,Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which isincorporated herein by reference in its entirety.

Polypeptide: As used herein, the term “polypeptide” or “polypeptide ofinterest” refers to a polymer of amino acid residues typically joined bypeptide bonds that can be produced naturally (e.g., isolated orpurified) or synthetically.

Subject: As used herein, the term “subject” refers to any organism towhich a composition in accordance with the disclosure may beadministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and humans) and/orplants. In some embodiments, a subject may be a patient.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of a disease, disorder, and/or condition.

Targeting moiety: As used herein, a “targeting moiety” is a compound oragent that may target a nanoparticle to a particular cell, tissue,and/or organ type.

Therapeutic Agent: The term “therapeutic agent” refers to any agentthat, when administered to a subject, has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect.

Transfection: As used herein, the term “transfection” refers to methodsto introduce a species (e.g., a polynucleotide, such as a mRNA) into acell.

Treating: As used herein, the term “treating” refers to partially orcompletely alleviating, ameliorating, improving, relieving, delayingonset of, inhibiting progression of, reducing severity of, and/orreducing incidence of one or more symptoms or features of a particularinfection, disease, disorder, and/or condition. For example, “treating”cancer may refer to inhibiting survival, growth, and/or spread of atumor. Treatment may be administered to a subject who does not exhibitsigns of a disease, disorder, and/or condition and/or to a subject whoexhibits only early signs of a disease, disorder, and/or condition forthe purpose of decreasing the risk of developing pathology associatedwith the disease, disorder, and/or condition.

Preventing: As used herein, the term “preventing” refers to partially orcompletely inhibiting the onset of one or more symptoms or features of aparticular infection, disease, disorder, and/or condition.

Tumor: As used herein, a “tumor” is an abnormal growth of tissue,whether benign or malignant.

Unmodified: As used herein, “unmodified” refers to any substance,compound or molecule prior to being changed in any way. Unmodified may,but does not always, refer to the wild type or native form of abiomolecule. Molecules may undergo a series of modifications wherebyeach modified molecule may serve as the “unmodified” starting moleculefor a subsequent modification.

Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the disclosure described herein. Thescope of the present disclosure is not intended to be limited to theDescription below, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The disclosure includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Thedisclosure includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process. It is also noted that the term “comprising” isintended to be open and permits but does not require the inclusion ofadditional elements or steps. When the term “comprising” is used herein,the term “consisting of” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the disclosure, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

EXAMPLES

The disclosure will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the disclosure. It is understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and scope of the appended claims.

Example 1: Modified mRNA Encoding hEPO Elicits an Anti-Drug Antibody(ADA) Response in Non-Human Primates

In this example, modified mRNA (mmRNA) encoding human erythropoietin(hEPO), but lacking any miR binding sites, was administered tocynomolgus macaques in a four week study to examine expression of hEPOin the animals.

mmRNA encoding hEPO was formulated into MC3 lipid nanoparticles (LNP),which include MC3 50%, DSPC 10%, Cholesterol 38.5%, PEG-DMG 1.5%,N:P˜5.5. (Values are based on mol. %). The mmRNA construct contained aCap 1 5′ Cap structure (7mG(5′)ppp(5′)NlmpNp), was fully modified with5-methylcytosine and 1-methylpseudouridine and comprised a 140nucleotide poly A tail. The mmRNA construct lacked the presence of anyinserted miR binding sites. The nucleotide sequence of thishEPO-encoding construct without any inserted miR binding sites is shownin SEQ ID NO: 7 (without the polyA tail shown).

The study comprised seven groups of animals. The negative control groupwas treated with PBS. Three treatment groups were treated with one ofthree different doses of the hEPO-encoding mmRNA LNP, at either 0.02mg/kg/dose, 0.1 mg/kg/dose or 0.2 mg/kg/dose. The low dose was selectedto achieve at least 2 times the therapeutically effective exposure forhuman EPO, whereas the high dose was selected to achieve at least 10times the levels of hEPO exposure over the therapeutically effectivedose. The positive control group was treated with recombinant hEPOprotein at a dose of 6000 U/kg. Two other controls groups were treatedwith either LNP containing an irrelevant mmRNA (non-translating mRNAform) or with LNP alone. The dose schedule was once per week for fourconsecutive weeks (for a total of 5 doses) by IV infusion. Infusionswere for 60 minutes (IV) via pumps. The four week dosing period wasfollowed by a four-week recovery evaluation period (control and highdose only).

The levels of expression of hEPO in the macaques was measured by ELISAsix hours post infusion on each of the treatment days (days 1, 8, 15, 22and 29). The results are shown in FIG. 1 . The results demonstrate thatthe macaques treated with the low dose of mmRNA (0.02 mg/kg/dose)exhibited a therapeutically effective level of hEPO that was maintainedat approximately the same level throughout the four-week study. Incontrast, in the macaques treated with the mid- and high-doses of mmRNA(0.1 mg/kg/dose and 0.2 mg/kg/dose), EPO levels declined startingbetween days 8 and 15 of the study and remained low for the duration ofthe study. Reticulocyte counts revealed a persistent reticulocytopeniain the animals treated at the mid- and high-doses of mmRNA (0.1mg/kg/dose and 0.2 mg/kg/dose). Furthermore, histological analysisrevealed decreased hematopoiesis in the bone marrow of the animalstreated with the mid- or high-doses of mmRNA. The reticulocytopenia anddecreased hematopoiesis in the animals treated with the higher doses ofmmRNA suggested that an anti-drug antibody (ADA) response may havedeveloped over time in the animals.

To determine whether an ADA response against human EPO was present,ELISAs were performed on serum from the animals to detect the presenceof cynomolgus macaque anti-human EPO antibodies. Blood was collected byfemoral venipuncture and serum was collected at room temperature andallowed to clot for at least 30 minutes, followed by centrifugation for10 minutes in a refrigerated (4° C.) centrifuge at 2700 rpm. StandardELISA methods were used for the detection of anti-human EPO IgG in theserum. Samples were analyzed in duplicate.

A series of negative control results were used to establish a thresholdlevel, above which would constitute a “positive” result in the assay.Representative quantitative ELISA results for the negative controlsamples, which thereby were used to set a threshold level, are shownbelow in Table 3:

TABLE 3 Results for the Anti- human EPO Antibody Analysis Negativecontrol (NC) results Assay ID: hAB Prod-01_(a) hAB Prod-02_(a) hABProd-03 Replicate A_(450 nm) A_(450 nm) A_(450 nm) 1 0.345 0.440 0.364 20.406 0.392 0.351 3 0.360 0.412 0.353 4 0.427 0.395 0.309 5 0.391 0.3900.325 6 0.402 0.398 0.364 7 0.397 0.377 0.368 8 0.401 0.379 0.353 90.392 0.429 0.326 10 0.392 0.376 0.345 11 0.412 0.375 0.358 12 0.3860.378 0.353 Mean 0.393 0.395 0.347 SD 0.022 0.022 0.018 % CV 5.6 5.5 5.2n 12 12 12 _(a)Results accepted in deviation, refer to Positive ControlSamples for the anti-human EPO antibody analysis.

The ELISA results for the seven different animal groups used in thestudy are shown below in Table 4:

TABLE 4 Cynomolgus Macaque Anti-Human EPO Antibody Response mmRNA mmRNAmmRNA Recomb. Animal hEPO hEPO hEPO hEPO Empty LNP NTX in LNP Number PBS0.02 mg/kg 0.1 mg/kg 0.2 mg/kg 6000 U/kg 0.2 mg/kg 0.2 mg/kg 1 − − + + −− − 2 − − − + +++ − − 3 − − + + ++ SI.+ − 4 − − − + +++ − − 5 − + + ++++ 6 − − − + +++ 7 − + 8 SI.+ ++ 9 − ++ 10 − ++ Total 1/10 1/6 3/6 10/105/6 1/4 0/4

The results demonstrate that all of the cynomolgus monkeys treated withthe highest dose of hEPO-encoding mmRNA (0.2 mg/kg/dose), and 50% of thecynomolgus monkeys treated with the mid-level dose (0.1 mg/kg/dose)exhibited an ADA response against human EPO. This was an unexpectedfinding and led the inventors to design and test additional mmRNAconstructs to attempt to reduce the ADA response to the encoded proteinin non-human primates. The results of those studies are described inExample 2.

Example 2: Incorporation of an miR-142-3p Binding Site into mmRNAInhibits an ADA Response to the Encoded Protein

In this example, a human EPO-encoding mmRNA construct was prepared thatincorporated an miR-142-3p binding site into the 3′ UTR of theconstruct. The mmRNA construct comprised a Cap 1 5′ cap(7mG(5′)ppp(5′)NlmpNp), was fully modified with 5-methylcytosine and1-methylpseudouridine and comprised a polyA tail of approximately 140nucleotides. A schematic diagram of the construct is shown in FIG. 2 .The nucleotide sequence of this human EPO-encoding mmRNA is shown in SEQID NO: 1 (without the poly A tail). The nucleotide sequence of the 3′UTR comprising the miR-142-3p binding site is shown in SEQ ID NO: 2. Thenucleotide sequence of the miR-142-3p binding site is shown in SEQ IDNO: 3. Other than the addition of the miR-142-3p binding site, the mmRNAconstruct and the LNP preparation were the same as in Example 1.

Cynomolgus macaque monkeys were treated with the construct comprisinghEPO-encoding mmRNA with the miR-142-3p binding site in the LNP at adose of 0.2 mg/kg/day. This dose previously had been shown to elicit anADA response against hEPO using a construct lacking the miR-142-3pbinding site (see Example 1). A four week study was conducted, in whichanimals were treated on days 1, 8, 15, 22 and 29 as described inExample 1. Intravenous infusions (60 minutes) were given via a temporaryindwelling catheter into the brachial or saphenous vein.

To examine whether an ADA response against hEPO was elicited in theanimals, ELISAs were performed on serum from the animals, as describedin Example 1, to detect the presence of cynomolgus macaque anti-humanEPO antibodies.

The results for the four animals (1301, 1302, 1303 and 1304), at each ofthe five time points for treatment at a dose of 0.2/mg/kg, are shownbelow in Table 5. The status of the sample as being “negative” or“positive” was determined relative a threshold value set based on theresults for negative control samples, as described in Example 1.

TABLE 5 Cynomolgus Macaque Anti-Human EPO Antibody Response MeanA450_(nm) Status Sample ID Time Point Value (Pos/Neg) 1301 Day 1 predose0.254 Negative Day 8 predose 0.255 Negative Day 15 predose 0.255Negative Day 22 predose 0.229 Negative Day 29 predose 0.230 Negative1302 Day 1 predose 0.107 Negative Day 8 predose 0.115 Negative Day 15predose 0.174 Negative Day 22 predose 0.243 Negative Day 29 predose0.282 Negative 1303 Day 1 predose 0.224 Negative Day 8 predose 0.244Negative Day 15 predose 0.485 Positive Day 22 predose 1 Positive Day 29predose 0.991 Positive 1304 Day 1 predose 0.142 Negative Day 8 predose0.162 Negative Day 15 predose 0.189 Negative Day 22 predose 0.1287Negative Day 29 predose 0.379 Negative

The results showed that only one of the four treated monkeys (1303)exhibited an ADA response against hEPO. Thus, when the miR-142-3pbinding site was incorporated into the hEPO-encoding mmRNA construct,only 25% of the animals treated with the high dose (0.2 mg/kg) of theconstruct exhibited an ADA against the encoded protein (Table 5),whereas treatment with the same dose using a construct that lacked themiR-142.p3 binding site resulted in 100% of the animals exhibiting anADA against the encoded protein (Table 4).

FIG. 3 shows a comparison of the ELISA results for the monkeys treatedwith 0.2 mg/kg of the mRNA construct lacking the miR-142-3p binding siteand the ELISA results for the monkeys treated with 0.2 mg/kg of the mRNAconstruct containing the miR-142-3p binding site, showing the levels ofanti-hEPO antibodies in the animals over time. Animals exhibiting ananti-drug-antibody (ADA) response are indicated. The results clearlydemonstrate that significantly more ADA responses were observed in theanimals treated with the construct lacking the miR binding site ascompared to the construct containing the miR binding site.

In a follow-up study in cynomolgus macaque monkeys, very similar resultsto the pilot study described above were observed. Administration of anhEPO-encoding mmRNA construct lacking the miR-142.p3 binding siteresulted in 100% of the animals exhibiting an ADA against the encodedprotein, whereas incorporation of a miR-142-3p binding site into theconstruct resulted in only 50% of the animals exhibiting an ADA againstthe encoded protein. Incorporation of three miR-142-3p binding sitesinto the construct did not lead to enhanced effects, therebydemonstrating that a single miR-142-3p binding site was sufficient forreduction of ADA against the encoded protein.

Accordingly, these results demonstrate that incorporation of amiR-142-3p binding site into the mmRNA construct is effective inreducing or eliminating an ADA response against the encoded protein.

Example 3: Incorporation of an miR-126 or miR-142 Binding Site intommRNA Inhibits B Cell Activation and Cytokine Expression

In this example, human EPO-encoding mmRNA constructs were prepared thatincorporated either a miR-142-3p binding site or a miR-126-3p bindingsite, or both the miR-142-3p and miR-126-3p binding sites, into the 3′UTR of the construct. The mmRNA constructs were administered to mice toexamine the effects of incorporating the miR binding sites on variousimmune parameters in the mice.

The mmRNA constructs contained a Cap 1 5′ Cap structure(7mG(5′)ppp(5′)NlmpNp), were fully modified with 5-methylcytosine and1-methylpseudouridine and comprised a 140 nucleotide poly A tail. Thecontrol mmRNA construct lacked the presence of any inserted miR bindingsites. The nucleotide sequence of this control hEPO-encoding constructwithout any inserted miR binding sites is shown in SEQ ID NO: 7 (withoutthe polyA tail shown). The nucleotide sequence of the hEPO-encodingconstruct with the inserted miR-142-3p binding site is shown in SEQ IDNO: 1 (without the polyA tail shown). The nucleotide sequence of thehEPO-encoding construct with the inserted miR-126-3p site is shown inSEQ ID NO: 28 (without the polyA tail shown). The nucleotide sequence ofthe hEPO-encoding construct with the inserted miR-142-3p and miR-126-3psites is shown in SEQ ID NO: 29 (without the polyA tail shown). Aschematic diagram of exemplary mmRNA constructs is shown in FIG. 2 . ThemmRNA constructs encoding hEPO were formulated into MC3 lipidnanoparticles (LNP), which include MC3 50%, DSPC 10%, Cholesterol 38.5%,PEG-DMG 1.5%, N:P˜5.5. (Values are based on mol. %).

In an initial study, groups of 30 mice each were assigned to thefollowing treatment groups: (i) hEpo construct without any miR bindingsites; (ii) hEpo construct with miR-142-3p binding site; (iii) hEpoconstruct with miR-126-3p binding site; and (iv) hEpo construct withmiR-142-3p and miR-126-3p binding sites. The mmRNAs were administeredintravenously to C57Bl/6 mice at a dose of 0.05 mg/kg. The dosingregimen was Days 1, 8, 15, 22, 29 and 36. For each treatment group, 5mice per group were dosed on each dosing day for a total of 6 doses. Theadditional 25 mice in each group were broken into 5 subsets such that 5mice were dosed once on each of dosing days 8, 15, 22, 29 and 36, for atotal of one dose per mouse.

Following treatment, mice were examined for: (i) protein expression fromthe mmRNA construct by measuring hEpo (in ng/ml), (ii) B cell frequencyby measuring % of splenic CD19+ cells, and (iii) activated B cellfrequency by measuring % of activated CD19+ cells in splenic CD19+cells. The results for these three read-outs are shown in FIGS. 4A, Band C, respectively. The results from this initial study demonstratedthat there was no noticeable difference in protein expression byinclusion of the miR-142-3p or miR-126 binding sites, alone or incombination, but there was a small but statistically significantdecrease in B cell activation observed by inclusion of the miR-126-3pbinding site, alone or in combination with the miR-142-3p binding sitein the mmRNA constructs.

To study this initial observation further, a second set of experimentswas conducted to test the mmRNA constructs at a higher dose (to providethe most sensitivity) and with a higher number of mice per group (toincrease the statistical confidence). The dosage regimen was Days 1 and8. Twelve mice per group were dosed intravenously on Day 1 with either0.2 mg/kg or 1 mg/kg of one of the four different mmRNA constructsdescribed above (hEpo; hEpo+miR-142-3p; hEPO+miR-126-3p; orhEpo+miR-142-3p/miR-126-3p), formulated in the MC3 LNP. At six hourspost-dosing on Day 1, serum was collected for analysis of protein levelexpression by Epo ELISA. Also, at six hours post-dosing on Day 1,spleens were harvested from six of the mice for B cell analysis. Theremaining six mice per group were dosed again on Day 8, followed byserum collection and spleen harvesting at 6 hours post-dosing forfurther analysis.

The results for the protein expression are shown in FIGS. 5A-B (for onedose, at either 0.2 mg/kg in FIG. 5A or 1 mg/kg in FIG. 5B) and FIGS.6A-B (for two doses, at either 0.2 mg/kg for FIG. 6A or 1 mg/kg for FIG.6B). The results demonstrated that the level of expression of theprotein of interest encoded by the mmRNA construct is not significantlyaffected by the inclusion of the miR binding site(s) (miR-142-3p,miR-126-3p or both) in the construct. Protein expression was furthermonitored through week 6 of the study. Expression was reduced by week 4and expression at week 5 was reduced almost to baseline (data notshown). At week 6, almost all mice in the control group lackedexpression whereas inclusion of the miR binding site(s) in the mRNAconstruct (miR-142-3p, miR-126-3p or both) maintained significant levelsof expression (FIG. 29 ).

The results for the B cell frequency and the activated B cell frequencyfor the single dose treatment are shown in FIGS. 7 and 8 , respectively.The results for the B cell frequency and the activated B cell frequencyfor the two dose treatment are shown in FIGS. 9 and 10 , respectively.These results showed that overall B cell frequency was not significantlyaffected by the inclusion of the miRs in the mmRNA constructs, but thatB cell activation is slightly decreased in the presence of themiR-142-3p binding site and significantly reduced by the presence of themiR-126-3p binding site (alone or in combination with the miR-142-3pbinding site). B cell frequency and activated B cell frequency weremeasured weekly through week 6 of the study and similar results wereobserved throughout the course of the study, with total B cellfrequencies not being significantly affected by inclusion of the miRbinding site(s) but with activated B cell frequencies being inhibited bythe presence of either the miR-142-3p or miR-126-3p binding site alone,or both miR binding sites in combination.

The results for IL-6 expression for the mice treated with one dose areshown in FIGS. 11A-B (0.2 mg/kg for FIG. 11A and 1 mg/kg for FIG. 11B)and for mice treated with two doses are shown in FIGS. 12A-B (0.2 mg/kgfor FIG. 12A and 1 mg/kg for FIG. 12B). These results demonstrate thatIL-6 expression is decreased in the presence of the miR-142-3p bindingsite and reduced even further by the presence of the miR-126-3p bindingsite (alone or in combination with the miR-142-3p binding site).

Cytokine expression was further examined by measuring TNF-α and IFN-γlevels as well as IL-6 levels. The results for these three cytokines formice treated with two doses at 0.2 mg/kg are shown in FIGS. 13A (IL-6),13B (TNF-α) and 13C (IFN-γ) and for mice treated with two doses at 1mg/kg are shown in FIGS. 14A (IL-6), 14B (TNF-α) and 14C (IFN-γ). Theseresults demonstrate that expression of all three cytokines (IL-6, TNF-αand IFN-γ) is decreased in the presence of the miR-142-3p binding siteand reduced even further by the presence of the miR-126-3p binding site(alone or in combination with the miR-142-3p binding site).

Cytokine expression was further monitored weekly through week 6 of thestudy, which demonstrated that levels of all three cytokines in theserum, IL-6, IFN-γ and TNF-α, were significantly inhibited by thepresence of either the miR-142-3p or miR-126-3p binding site alone, orboth miR binding sites in combination, in the mRNA construct.

Thus, the studies described above demonstrate that incorporation into anmRNA construct of a miR-126-3p binding site, a miR-142-3p binding siteor both miR binding sites in combination, results in a reduced frequencyof B cell activation and in reduced secretion of a panel of differentimmune-stimulating cytokines (IL-6, TNF-α, IFN-γ) in vivo in animalstreated with the mRNA construct, while having a minimal impact on theexpression of a protein of interest encoded by the mRNA construct in thetreated animal.

Example 4: Additional Studies Incorporating miR-126 and/or miR-142Binding Sites into mmRNA Constructs

In this example, luciferase-encoding mmRNA constructs were prepared thatincorporated either a miR-142-3p binding site or a miR-126-3p bindingsite, or both the miR-142-3p and miR-126-3p binding sites, into the 3′UTR of the construct. The mmRNA constructs were administered to mice toexamine the effects of incorporating the miR binding sites on variousimmune parameters in the mice. The mmRNA constructs contained a Cap 1 5′Cap structure (7mG(5′)ppp(5′)NlmpNp), were fully modified with5-methylcytosine and 1-methylpseudouridine and comprised a 100nucleotide poly A tail. The control mmRNA construct lacked the presenceof any known miR binding sites. The mmRNA constructs encoding luciferase(Luc) were formulated into MC3 lipid nanoparticles (LNP), which includeMC3 50%, DSPC 10%, Cholesterol 38.5%, PEG-DMG 1.5%, N:P˜5.5. (Values arebased on mol. %).

In an initial study, groups of 24 mice each were assigned to thefollowing treatment groups: (i) Luciferase (Luc) construct without anymiR binding sites (the sequence of which is shown in SEQ ID NO: 30);(ii) Luc construct with miR-142-3p binding site (the sequence of whichis shown in SEQ ID NO: 31); (iii) Luc construct with miR-126-3p bindingsite (the sequence of which is shown in SEQ ID NO: 32); and (iv) Lucconstruct with miR-142-3p and miR-126-3p binding sites (the sequence ofwhich is shown in SEQ ID NO: 33). The mmRNAs were administeredintravenously to C57Bl/6 mice at a dose of 0.2 mg/kg. In each group, 15mice were selected to be dosed on days 1, 8, 15, 22, 29 and 36, 3 micewere dosed only once on Day 22, and 3 mice were dosed only once on Day36. From the 15 mice selected for repeat dosing, 5 each were sacrificedfor spleen collection 6 h after dose on Day 1, 6 h after dose on Day 22and 6 h after dose on Day 36.

Following treatment, mice were examined for: (i) luciferase expressionfrom the mmRNA construct by measuring whole body luminescence; (ii) Bcell frequency by measuring % of splenic CD19+ cells; (iii) activated Bcell frequency by measuring % of activated CD19+ cells in splenic CD19+cells; and (iv) cytokine production (IL-6, IFN-γ, TNF-α).

The results for the Luciferase expression are shown in FIGS. 15A and15B. FIG. 15A shows results after 1 week (1 dose) and FIG. 15B showsresults after 2 weeks (or 2 doses). The levels of Luc were much morevariable that the hEPO levels discussed in Example 1. However, theresults demonstrated that the level of expression of the protein ofinterest (Luc) encoded by the mmRNA construct is not significantlyaffected by the inclusion of the miR binding site(s) (miR-142-3p,miR-126-3p or both) in the construct. Protein expression was furthermonitored through week 6 of the study. Lucierfase expression showedmodest to no change through the 6 weeks. FIG. 30 shows expression atweek 5.

The results for the B cell frequency and the activated B cell frequencyare shown in FIGS. 16 and 17 , respectively. The results shown are atweek 1 (1 dose). These results showed that overall total B cellfrequency was not significantly affected by the inclusion of the miRbinding site(s) in the Luc mmRNA constructs, but that frequency ofactivated B cells is slightly decreased in the presence of themiR-142-3p binding site and significantly reduced by the presence of themiR-126-3p binding site (alone or in combination with the miR-142-3pbinding site). The reduced activated B cell frequency was not due toreduced numbers of CD19⁺ cells as (i) we measured the percentage ofactivated B cells inside the total B cell population, and (ii) the Bcell frequencies did not fluctuate between groups. B cell frequency andactivated B cell frequency were measured weekly through week 6 of thestudy and similar results were observed throughout the course of thestudy, with total B cell frequencies not being significantly affected byinclusion of the miR binding site(s) but with activated B cellfrequencies being inhibited by the presence of either the miR-142-3p ormiR-126-3p binding site alone, or both miR binding sites in combination.

Cytokine expression was examined by measuring IL-6, IFN-γ and TNF-αlevels in serum. The results for these three cytokines for mice treatedwith 0.2 mg/kg mmRNA are shown in FIGS. 18A (IL-6), 18B (IFN-γ) and 18C(TNF-α). The results shown are for week 2; similar results were observedat week 1 and week 3. Again, more variability in cytokine expression wasobserved in the Luc treated animals as compared to the hEPO treatedanimals described in Example 1. However, these results demonstrate thatexpression of at least some cytokines (most markedly with IFN-γ andTNF-α) is decreased in the presence of the miR-142-3p binding siteand/or the miR-126-3p binding site, alone or in combination. Cytokineexpression was further monitored through week 6 of the study, whichdemonstrated that levels of all three cytokines in the serum, IL-6,IFN-γ and TNF-α, were significantly inhibited by the presence of eitherthe miR-142-3p or miR-126-3p binding site alone, or both miR bindingsites in combination, in the mRNA construct.

Thus, the studies described above demonstrate that incorporation into anmRNA construct of a miR-126-3p binding site, a miR-142-3p binding siteor both miR binding sites in combination, results in a reduced frequencyof B cell activation and in reduced secretion of a panel of differentimmune-stimulating cytokines (TNF-α, IFN-γ) in vivo in animals treatedwith the mRNA construct, while having a minimal impact on the expressionof a protein of interest encoded by the mRNA construct in the treatedanimal.

Example 5: Additional Studies Incorporating miR-142 and/or miR-155Binding Sites into mmRNA Constructs

In this example, EPO-encoding mmRNA constructs were prepared thatincorporated a miR-142-3p binding site, a miR-142-5p binding site, amiR-155-5p binding site, or combinations thereof, and/or multiple copiesthereof, into the 3′ UTR of the construct. The miR-155-5p sequence uponwhich the binding site insertion was designed is as follows:uuaaugcuaauugugauaggggu (SEQ ID NO: 34). The miR-155-5p binding siteinserted into the 3′ UTR has the sequence as follows:ACCCCTATCACAATTAGCATTAA (SEQ ID NO: 35).

The mmRNA constructs were administered to mice to examine the effects ofincorporating the miR binding sites on various immune parameters in themice. The mmRNA constructs contained a Cap 1 5′ Cap structure(7mG(5′)ppp(5′)NlmpNp), were fully modified with 5-methylcytosine and1-methylpseudouridine and comprised a 100 nucleotide poly A tail. Thecontrol mmRNA construct lacked the presence of any inserted miR bindingsites. The mmRNA constructs encoding EPO were formulated into MC3 lipidnanoparticles (LNP), which include MC3 50%, DSPC 10%, Cholesterol 38.5%,PEG-DMG 1.5%, N:P˜5.5. (Values are based on mol. %).

In this study, mice were assigned to one of the following treatmentgroups: (i) EPO construct without any miR binding sites (the sequence ofwhich is shown in SEQ ID NO: 36); (ii) EPO construct with one miR-142-3pbinding site (the sequence of which is shown in SEQ ID NO: 37); (iii)EPO construct with three miR-142-3p binding sites (EPO_3X-miR-142-3p)(the sequence of which is shown in SEQ ID NO: 38); (iv) EPO constructwith one miR-142-5p binding site (the sequence of which is shown in SEQID NO: 39); (v) EPO construct with three miR-142-5p binding sites(EPO_3X-miR-142-5p) (the sequence of which is shown in SEQ ID NO: 40);(vi) EPO construct with two miR-142-5p binding sites and one miR-142-3pbinding sites (EPO_2X-miR-142-5p_1X-miR-142-3p) (the sequence of whichis shown in SEQ ID NO: 41); (vii) EPO construct with one miR-155-5pbinding site (the sequence of which is shown in SEQ ID NO: 42); (viii)EPO construct with three miR-155-5p binding sites (EPO_3X-miR-155) (thesequence of which is shown in SEQ ID NO: 43); (ix) EPO construct withtwo miR-155-5p binding sites and one miR-142-3p binding sites(EPO_2X-miR-155-5p_1X-miR-142-3p) (the sequence of which is shown in SEQID NO: 44); and (x) LNP with non-translating control sequence (“empty”).The mmRNAs were administered intravenously to C57Bl/6 mice at a dose of0.2 mg/kg. The dosing regimen was Days 1 and 8.

Following treatment, mice were examined for: (i) protein expression fromthe mmRNA construct by measuring levels of the encoded EPO (in ng/ml);(ii) total B cell frequency by measuring % of splenic CD19⁺ cells; (iii)activated B cell frequency by measuring % of activated CD19⁺ cells insplenic CD19⁺ cells; and (iv) cytokine production (IL-6, IFN-γ, TNF-α).

The results for the EPO protein expression are shown in FIGS. 19A-B,with FIG. 19A showing the results at week 1 and FIG. 19B showing theresults for week 2. The results demonstrated that the level ofexpression of the protein of interest (EPO) encoded by the mmRNAconstruct is not significantly affected by the inclusion of the miRbinding site(s) (miR-142-3p, miR-142-5p, miR-155-5p or multiple copiesand/or combinations thereof) in the construct.

The results for the B cell frequency and the activated B cell frequencyare shown in FIGS. 20 and 21 , respectively, for week 1, and FIGS. 22and 23 , respectively, for week 2. These results showed that overalltotal B cell frequency was not significantly affected by the inclusionof the miRs in the EPO mmRNA constructs, but that the frequency ofactivated B cells is decreased in the presence of the miR-142-3p bindingsite, the miR-142-5p binding site, the miR-155-5p binding site, ormultiple copies thereof and/or combinations thereof.

Cytokine expression was examined by measuring IL-6, TNF-α and IFN-γlevels in the serum. The results for these three cytokines for micetreated with 0.2 mg/kg mmRNA are shown in FIGS. 24A (IL-6), 24B (TNF-α)and 24C (IFN-γ). The results shown are for week 2. Variability incytokine expression was observed in the EPO treated animals. However,these results demonstrate that expression of at least some cytokines isdecreased in the presence of the miR-142-3p binding site, the miR-142-5pbinding site, the miR-155-5p binding site, or multiple copies thereofand/or combinations thereof.

Thus, the studies described above demonstrate that incorporation into anmRNA construct of a miR-142-3p binding site, a miR-142-5p binding site,a miR-155-5p binding site, or multiple copies thereof and/orcombinations thereof, results in a reduced frequency of B cellactivation and in reduced secretion of a panel of differentimmune-stimulating cytokines in vivo in animals treated with the mRNAconstruct, while having a minimal impact on the expression of a proteinof interest encoded by the mRNA construct in the treated animal.

Example 6: Effect of miR Binding Sites on Particular Immune CellPopulations

In this example, studies were performed to examine the effect of theinclusion in mRNA constructs of a miR binding site expressed in immunecells (miR-142, miR-126 or both miR-142+miR-126) on the frequency andactivation of particular immune cell populations. Balb/c mice weretreated with mRNA constructs encoding EPO as described above in Example3, which constructs either lacked miR binding sites or contained eithera miR-142-3p binding site, a miR-126-3p binding site or both amiR-142-3p binding site and a miR-126-3p binding site. Mice were treatedintravenously on days 1, 8 and 15 with 0.2 mg/kg of mRNA constructformulated into MC3 lipid nanoparticles.

A first set of experiments examined whether the miRs had any effect onthe frequency of CD27⁺ B cells or the level of CD27 expression in thesecells. The results are shown in FIGS. 25A-B. FIG. 25A shows thefrequency of CD27⁺ CD19⁺ B cells in splenic CD19⁺ B cells, demonstratingthat the CD27⁺ B cell population was not affected by the inclusion ofthe miR binding site(s) in the mRNA constructs. FIG. 25B shows the levelof CD27 expression in the CD27⁺ B cell population and, likewise, showsthat this expression was not affected by the inclusion of the miRbinding site(s) in the mRNA constructs. Thus, this first set ofexperiments demonstrated that the effect of the miR binding site(s) ininhibiting B cell activation and/or inhibiting cytokine production wasnot resulting from the miR binding site(s) affecting either thefrequency of CD27⁺ CD19⁺ B cells or the level of CD27 expression inthese cells.

A second set of experiments examined whether the miRs had any effect onthe frequency of CD11c⁺ cells, as a marker of dendritic cells, and/orthe frequency of activated dendritic cells (CD11c⁺ CD70⁺ CD86⁺ cells).The results are shown in FIGS. 26A-B. FIG. 26A shows the percentage ofCD11c⁺ cells in splenic cells from the mice, demonstrating that thetotal frequency of CD11c⁺ cells was inhibited by the inclusion of themiR-142-3p binding site, the miR-126-3p binding site, or both bindingsites, in the mRNA constructs. FIG. 26B shows the frequency of activateddendritic cells (CD11c⁺ CD70⁺ CD86⁺ cells) within the CD11c⁺ spleniccell population and, likewise, shows that the frequency of activateddendritic cells was inhibited by the inclusion of the miR bindingsite(s) in the mRNA constructs. Thus, this second set of experimentsdemonstrated that the inclusion of the miR binding site(s) inhibitedboth the total frequency of dendritic cells and the frequency ofactivated dendritic cells, thereby implicating this cell population inthe mechanism of how inclusion of the miR binding site(s) leads toinhibition of B cell activation and inhibition of cytokine production.While not intending to be limited by mechanism, these results suggestthat in the mice, decreased levels of activated CD70⁺ dendritic cellsleads to decreased interaction with CD27⁺ B cells, thereby leading todecreased B cell activation and decreased cytokine production.

To further investigate this, a third set of experiments were performedin which the ability of plasmacytoid dendritic cells (pDCs) from themice to stimulate proliferation of naïve B cells was examined. For theseexperiments, 2×10⁵ naïve B cells labeled with CFSE were incubated with4×10⁴ pDCs purified from the spleens of mice injected either with hEPOmRNA or hEPO mRNA-miR142, hEPO mRNA-miR126 or hEPO mRNA-miR126+142 inthe presence of anti IgM at 10 After 5 days of culture, cells wereharvested and analyzed by flow cytometry for CFSE expression. Levels ofproliferation of the naïve B cells incubated with the pDCs are shown inthe graph of FIG. 27 . The results demonstrate that naïve B cellsproliferate less in the presence of pDCs from mice treated with the mRNAconstructs including the miR-142 binding site, the miR-126 binding siteor both binding sites. These experiments further support the mechanismthat in mice treated with the miR-containing constructs, decreasedlevels of activated pDCs leads to decreased B cell stimulation, therebyleading to inhibition of B cell activation and inhibition of cytokineproduction.

Example 7: Effect of miR Binding Sites on IgM

In this example, studies were performed to examine the effect of theinclusion in mRNA constructs of a miR binding site expressed in immunecells (miR-142, miR-126 or both miR-142+miR-126) on the levels of IgMthat binds to PEG (anti-PEG IgM). Since PEG is a component of the LNP inwhich the mRNA construct is encapsulated, the presence of anti-PEG IgMin the serum of the mice contributes to Accelerated Blood Clearance(ABC) of the LNP/mRNA composition.

Mice were treated with the Luciferase-encoding mRNA constructs asdescribed in Example 2. Serum was collected from the mice after thesecond, third and fourth doses of treatment and the levels of anti-PEGIgM in the serum (in ng/ml) was measured. The results are shown in thegraphs of FIGS. 28A-C. FIG. 28A shows anti-PEG IgM levels after thesecond dose, FIG. 28B shows anti-PEG IgM levels after the third dose andFIG. 28C shows anti-PEG IgM levels after the fourth dose. The resultsdemonstrate that inclusion of the miR binding site(s) significantlydecreased the level of anti-PEG IgM in the mice. This effect was alreadyobserved after treatment with the second dose and the effect continuedthrough treatment with the fourth dose. This reduction of anti-PEG IgMin the mice treated with the miR binding site-containing mRNA constructsindicates that ABC, which is mediated at least in part by anti-PEG IgM,is expected to be reduced in these mice.

Example 8: Effect of Single Versus Multiple miR Binding Sites

In this example, studies were performed to examine the effect of theinclusion in mRNA constructs of a single miR binding site (1×) versusmultiple miR binding sites (e.g., 3×, having 3 miR binding sites).

A first series of studies were performed examining miR-122, which isknown to regulate mRNA expression in liver cells. To determine theeffect of inclusion of one versus three miR-122 binding sites onexpression of a protein encoded by an mRNA construct, primaryhepatocytes were co-transfected using Lipofectamine 2000 with aluciferase mRNA construct (Luc) and an enhanced green fluorescentprotein mRNA construct (eGFP), wherein the 3′ UTR of each constructeither (i) lacked any miR-122 binding sites (control); (ii) containedone miR-122 binding site; or (iii) contained three miR-122 bindingsites. Another control included transfection with Luc-like and eGFP-likeRNA sequences with no ATG in the putatative coding sequence. The mRNAsequences of these four eGFP constructs are shown in SEQ ID NOs: 61-64,respectively. The mRNA sequences of these four Luc constructs are shownin SEQ ID NOs: 65-68, respectively. The sequence of the 3′ UTRcontaining three miR-122 bindings sites is shown in SEQ ID NO: 54.

Cells were transfected with either 7.5 ng, 15 ng, 50 ng or 100 ng ofmRNA. Total green integrated intensity was measured over time for 24hours. The results are shown in FIGS. 31A-D, which demonstrate that atlower mRNA doses (7.5 ng and 15 ng, in FIGS. 31A and 31B, respectively),both the 1×miR-122 and the 3×miR-122 constructs led to significantreduction in expression of eGFP in the primary hepatocytes, whereas atthe higher mRNA doses (50 ng and 100 ng, in FIGS. 31C and 31D,respectively), the 3×miR-122 constructs exhibited greater inhibition ofeGFP expression than the 1×miR-122 constructs, and at the higher dosesthe 3×miR-122 constructs were able to maintain that loss of proteinexpression over time. Luciferase luminescence was also examined in theprimary hepatocytes and similar results were observed: that the3×miR-122 constructs led to higher knock-down of protein expression,particularly at higher mRNA doses.

In a second series of experiments examining the effect of inclusion ofone versus three miR-122 binding sites on expression of a proteinencoded by an mRNA construct, primary hepatocytes were transfected usingLipofectamine 2000 with an mRNA construct encoding a caspase and havingin its 3′ UTR either (i) no miR-122 binding sites (control); (ii) onemiR-122 binding site; or (iii) three miR-122 binding sites. Anothercontrol included transfection with Caspase-like RNA sequences with noATG in the putatative coding sequence. The mRNA sequences of these fourcaspase constructs are shown in SEQ ID NOs: 69-72, respectively.Caspase-mediated toxicity was measured over time for 24 hours. Theresults are shown in FIGS. 32A-D, which demonstrate that at the lowermRNA dose (7.5 ng, in FIG. 32A), both the 1×miR-122 and the 3×miR-122constructs inhibited caspase-mediated toxicity, with the 3×miR-122construct having a much stronger effect in inhibiting toxicity.Moreover, at the higher mRNA doses (15 ng, 50 ng and 100 ng, in FIGS.32B, 32C and 32D, respectively), only the 3×miR-122 construct was ableto inhibit caspase-mediated toxicity. Studies were also performedtransfecting primary hepatocytes with the caspase mRNA construct in anMC3 lipid nanoparticle and similar results were observed: thealleviation of caspase-mediated toxicity was significantly stronger withthe 3×miR-122 construct than with the 1×miR-122 construct.

To confirm these in vitro study results in vivo, Balb/c mice wereco-adminstered the Luc and eGFP mRNA constructs (0.5 mg/kg), in MC3nanoparticles, containing either zero, one or three miR-122 bindingsites. eGFP fluorescence in the liver was examined, the results of whichshowed that inclusion of one miR-122 binding site led to modestknock-down of eGFP expression in the liver, while inclusion of threemiR-122 binding sites led to significant knock-down of eGFP expressionin the liver. Similar results were observed for luciferase expression,with 1× site reducing luciferase expression and 3× sites leading togreater knock-down of protein expression.

In a third series of experiments, cynomolgus monkeys were administeredhEPO mRNA constructs (0.2 mg/kg), in MC3 nanoparticles, containingeither zero, one or three miR-142-3p binding sites in the 3′ UTR. Thelevels of expression of hEPO (in ng/ml) in the monkeys was measured byELISA, the results of which are shown in FIG. 33 . The results show thatinclusion of 3×miR-142-3p binding sites in the hEPO construct led tosignificantly lower expression of hEPO compared to the constructs withzero or one miR-142-3p binding site.

Overall, the studies described above demonstrate the benefit ofinclusion of at least one miR binding site in the mRNA constructs andthe enhanced effect of inclusion of three miR binding sites in theconstruct.

Example 9: Effect of miR Binding Sites in the 5′ Untranslated Region (5′UTR)

In this example, studies were performed to examine the effect of theinclusion in mRNA constructs of a miR binding site in one of threedifferent insertion sites within the 5′ UTR of the mRNA construct. The5′UTR sequence used is shown below: GGGAAATAAGAG{circumflex over( )}AGAAAAGAAGAGTA{circumflex over ( )}AGAAGAAATATA{circumflex over( )}AGAGCCACC (SEQ ID NO: 53), with the three possible insertions sites(P1, P2, P3) indicated by a caret sign ({circumflex over ( )}). Thesequences of 5′ UTRs having a miR-142-3p binding site inserted intoeither P1, P2 or P3 are shown in SEQ ID NOs: 55-57, respectively. Thesequences of 5′ UTRs having a miR-122 binding site inserted into eitherP1, P2 or P3 are shown in SEQ ID NOs: 58-60, respectively.

In a first series of experiments, enhanced green fluorescent protein(eGFP) mRNA constructs were transfected into RAW264.7 murine macrophagecells using Lipofectamine 2000, wherein the constructs contained either1× or 3×miR-142-3p binding sites in the 3′ UTR, or contained amiR-142-3p binding site inserted into either P1, P2 or P3 of the 5′ UTR,or contained a miR-142-3p binding site inserted into either P1, P2 or P3of the 5′ UTR combined with a single miR-142-3p binding site in the 3′UTR. eGFP fluorescence was measured at 48 hours post-transfection. Theresults are shown in FIG. 34 . The results demonstrate that all of theconstructs tested, including those with the miR-142-3p binding siteinserted at P1, P2 or P3, alone or in combination with a miR-142-3pbinding site in the 3′ UTR, led to significantly reduced proteinexpression in the cells.

Similar constructs were made using hEPO as the encoded protein andsimilar experiments were conducted with transfected RAW264.7 cells. Theresults with the hEPO constructs were very similar to those with theeGFP constructs, with all constructs tested, including those with themiR binding site inserted at P1, P2 or P3, alone or in combination witha miR binding site in the 3′ UTR, leading to significantly reducedprotein expression in the cells. With the hEPO constructs, however, thedegree of inhibition by the P1, P2 and P3 5′ UTR constructs was slightlyless (60%, 73% and 93%, respectively) compared to the degree ofinhibition with the eGFP constructs (89%, 96% and 98%, respectively).

In a second series of experiments, hEPO mRNA constructs were madesimilar to those described above, except that the constructs containedeither 1× or 3×miR-122 binding sites in the 3′ UTR, or contained amiR-122 binding site inserted into either P1, P2 or P3 of the 5′ UTR, orcontained a miR-122 binding site inserted into either P1, P2 or P3 ofthe 5′ UTR combined with a single miR-122 binding site in the 3′ UTR.The mRNA constructs were transfected into primary hepatocytes usingLipofectamine 2000. hEPO expression (in ng/mL) was measured by ELISA,the results of which are shown in FIG. 35 . The results demonstrate thatall of the constructs tested, including those with the miR-122 bindingsite inserted at P1, P2 or P3, alone or in combination with a miR-122binding site in the 3′ UTR, led to significantly reduced proteinexpression in the cells.

To confirm these in vitro study results in vivo, Balb/c mice wereadministered the panel of hEPO/miR-122 mRNA constructs in MC3nanoparticles or were administered the panel of hEPO/miR-142-3pconstructs using Lipofectamine 2000. The results showed that, similar tothe in vitro observations, all of the constructs tested, including thosewith a miR-122 or miR-142-3p binding site inserted at P1, P2 or P3,alone or in combination with a miR-122 or miR-142-3p binding site in the3′ UTR, led to significantly reduced protein expression in the mice.

Overall, these studies demonstrate the benefit of inclusion of at leastone miR binding site in either the 3′ UTR or the 5′ UTR of the mRNAconstruct, or a combination of miR binding sites in both the ‘3 UTR andthe 5’ UTR.

SUMMARY OF SEQUENCE LISTING SEQ ID NO: Sequence 1GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGGAGUGCACGAGUGUCCCGCGUGGUUGUGGUUGCUGCUGUCGCUCUUGAGCCUCCCACUGGGACUGCCUGUGCUGGGGGCACCACCCAGAUUGAUCUGCGACUCACGGGUACUUGAGAGGUACCUUCUUGAAGCCAAAGAAGCCGAAAACAUCACAACCGGAUGCGCCGAGCACUGCUCCCUCAAUGAGAACAUUACUGUACCGGAUACAAAGGUCAAUUUCUAUGCAUGGAAGAGAAUGGAAGUAGGACAGCAGGCCGUCGAAGUGUGGCAGGGGCUCGCGCUUUUGUCGGAGGCGGUGUUGCGGGGUCAGGCCCUCCUCGUCAACUCAUCACAGCCGUGGGAGCCCCUCCAACUUCAUGUCGAUAAAGCGGUGUCGGGGCUCCGCAGCUUGACGACGUUGCUUCGGGCUCUGGGCGCACAAAAGGAGGCUAUUUCGCCGCCUGACGCGGCCUCCGCGGCACCCCUCCGAACGAUCACCGCGGACACGUUUAGGAAGCUUUUUAGAGUGUACAGCAAUUUCCUCCGCGGAAAGCUGAAAUUGUAUACUGGUGAAGCGUGUAGGACAGGGGAUCGCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (EPO with miR 142-3p binding site) 2GCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3′UTR with miR 142-3p binding site) 3UCCAUAAAGUAGGAAACACUACA (miR 142-3p binding site) 4GSGATNFSLLKQAGDVEENPGP (2A peptide) 5GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAAC CCTGGACCT(polynucleotide encoding 2A peptide) 6TCCGGACTCAGATCCGGGGATCTCAAAATTGTCGCTCCTGTCAAACAAACTCTTAACTTTGATTTACTCAAACTGGCTGGGGATGTAGAAAGCAATCCAGGTCCACTC(polynucleotide encoding 2A peptide) 7GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGGAGUGCACGAGUGUCCCGCGUGGUUGUGGUUGCUGCUGUCGCUCUUGAGCCUCCCACUGGGACUGCCUGUGCUGGGGGCACCACCCAGAUUGAUCUGCGACUCACGGGUACUUGAGAGGUACCUUCUUGAAGCCAAAGAAGCCGAAAACAUCACAACCGGAUGCGCCGAGCACUGCUCCCUCAAUGAGAACAUUACUGUACCGGAUACAAAGGUCAAUUUCUAUGCAUGGAAGAGAAUGGAAGUAGGACAGCAGGCCGUCGAAGUGUGGCAGGGGCUCGCGCUUUUGUCGGAGGCGGUGUUGCGGGGUCAGGCCCUCCUCGUCAACUCAUCACAGCCGUGGGAGCCCCUCCAACUUCAUGUCGAUAAAGCGGUGUCGGGGCUCCGCAGCUUGACGACGUUGCUUCGGGCUCUGGGCGCACAAAAGGAGGCUAUUUCGCCGCCUGACGCGGCCUCCGCGGCACCCCUCCGAACGAUCACCGCGGACACGUUUAGGAAGCUUUUUAGAGUGUACAGCAAUUUCCUCCGCGGAAAGCUGAAAUUGUAUACUGGUGAAGCGUGUAGGACAGGGGAUCGCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGG CGGC(human EPO no miR binding sites) 8 UGUAGUGUUUCCUACUUUAUGGA(miR 142-3p sequence) 9 CAUAAAGUAGAAAGCACUACU (miR 142-5p sequence) 10CCUCUGAAAUUCAGUUCUUCAG (miR 146-3p sequence) 11 UGAGAACUGAAUUCCAUGGGUU(miR 146-5p sequence) 12 CUCCUACAUAUUAGCAUUAACA (miR 155-3p sequence) 13UUAAUGCUAAUCGUGAUAGGGGU (miR 155-5p sequence) 14 UCGUACCGUGAGUAAUAAUGCG(miR 126-3p sequence) 15 CAUUAUUACUUUUGGUACGCG (miR 126-5p sequence) 16CCAGUAUUAACUGUGCUGCUGA (miR 16-3p sequence) 17 UAGCAGCACGUAAAUAUUGGCG(miR 16-5p sequence) 18 CAACACCAGUCGAUGGGCUGU (miR21-3p sequence) 19UAGCUUAUCAGACUGAUGUUGA (miR21-5p sequence) 20 UGUCAGUUUGUCAAAUACCCCA(miR 223-3p sequence) 21 CGUGUAUUUGACAAGCUGAGUU (miR 223-5p sequence) 22UGGCUCAGUUCAGCAGGAACAG (miR 24-3p sequence) 23 UGCCUACUGAGCUGAUAUCAGU(miR 24-5p sequence) 24 UUCACAGUGGCUAAGUUCCGC (miR27-3p sequence) 25AGGGCUUAGCUGCUUGUGAGCA (miR27-5p sequence) 26 CGCAUUAUUACUCACGGUACGA(miR 126-3p binding site) 27UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC

UCUUUGAAUAAAGUCUGAGUGGGCGGC (3′UTR with miR 126-3p binding site) 28GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGGAGUGCACGAGUGUCCCGCGUGGUUGUGGUUGCUGCUGUCGCUCUUGAGCCUCCCACUGGGACUGCCUGUGCUGGGGGCACCACCCAGAUUGAUCUGCGACUCACGGGUACUUGAGAGGUACCUUCUUGAAGCCAAAGAAGCCGAAAACAUCACAACCGGAUGCGCCGAGCACUGCUCCCUCAAUGAGAACAUUACUGUACCGGAUACAAAGGUCAAUUUCUAUGCAUGGAAGAGAAUGGAAGUAGGACAGCAGGCCGUCGAAGUGUGGCAGGGGCUCGCGCUUUUGUCGGAGGCGGUGUUGCGGGGUCAGGCCCUCCUCGUCAACUCAUCACAGCCGUGGGAGCCCCUCCAACUUCAUGUCGAUAAAGCGGUGUCGGGGCUCCGCAGCUUGACGACGUUGCUUCGGGCUCUGGGCGCACAAAAGGAGGCUAUUUCGCCGCCUGACGCGGCCUCCGCGGCACCCCUCCGAACGAUCACCGCGGACACGUUUAGGAAGCUUUUUAGAGUGUACAGCAAUUUCCUCCGCGGAAAGCUGAAAUUGUAUACUGGUGAAGCGUGUAGGACAGGGGAUCGCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC

GUGGU CUUUGAAUAAAGUCUGAGUGGGCGGC (hEPO with miR 126-3p binding site) 29GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGGAGUGCACGAGUGUCCCGCGUGGUUGUGGUUGCUGCUGUCGCUCUUGAGCCUCCCACUGGGACUGCCUGUGCUGGGGGCACCACCCAGAUUGAUCUGCGACUCACGGGUACUUGAGAGGUACCUUCUUGAAGCCAAAGAAGCCGAAAACAUCACAACCGGAUGCGCCGAGCACUGCUCCCUCAAUGAGAACAUUACUGUACCGGAUACAAAGGUCAAUUUCUAUGCAUGGAAGAGAAUGGAAGUAGGACAGCAGGCCGUCGAAGUGUGGCAGGGGCUCGCGCUUUUGUCGGAGGCGGUGUUGCGGGGUCAGGCCCUCCUCGUCAACUCAUCACAGCCGUGGGAGCCCCUCCAACUUCAUGUCGAUAAAGCGGUGUCGGGGCUCCGCAGCUUGACGACGUUGCUUCGGGCUCUGGGCGCACAAAAGGAGGCUAUUUCGCCGCCUGACGCGGCCUCCGCGGCACCCCUCCGAACGAUCACCGCGGACACGUUUAGGAAGCUUUUUAGAGUGUACAGCAAUUUCCUCCGCGGAAAGCUGAAAUUGUAUACUGGUGAAGCGUGUAGGACAGGGGAUCGCUGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC

GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(hEPO with miR 142-3p and miR 126-3p binding sites) 30TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGG GCGGC(3′ UTR, no miR binding sites) 31TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCTCCATAAAGTAGGAAACACTACAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (3′ UTR with miR 142-3p binding site) 32TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCC

GTGG TCTTTGAATAAAGTCTGAGTGGGCGGC (3′ UTR with miR 126-3p binding site)33 TGATAATAG TCCATAAAGTAGGAAACACTACAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCC

GTGGTCTTTGAATAAAGTCTGAGTGGGCGGC(3′ UTR with miR 142-3p and miR 126-3p binding sites) 34UUAAUGCUAAUUGUGAUAGGGGU (miR 155-5p sequence) 35 ACCCCTATCACAATTAGCATTAA(miR 155-5p binding site) 36TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGG GCGGC(3′UTR with no miR binding site) 37UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCATAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3′UTR with miR 142-3p binding site) 38TGATAATAG TCCATAAAGTAGGAAACACTACAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCATAAAGTAGGAAACACTACATCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCTCCATAAAGTAGGAAACACTACAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (3′ UTR with 3 miR 142-3p binding sites) 39TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCC

GTGGT CTTTGAATAAAGTCTGAGTGGGCGGC (3′UTR with miR 142-5p binding site) 40

GCTGGAGCCTCGGTGGCCATGCTTCTT GCCCCTTGGGCC

TCCCCCCAGCCCCTCCTCCCCTTC CTGCACCCGTACCCCC

GTGGTCTTTGAATAAAGTCT GAGTGGGCGGC (3′UTR with 3 miR 142-5p binding sites)41

GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCATAAAGTAGGAAACACTACATCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCC

GTGGTCTTTGAATAAAGT CTGAGTGGGCGGC(3′UTR with 2 miR 142-5p binding sites and 1 miR 142-3p binding site) 42TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCACCCCTATCACAATTAGCATTAAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (3′UTR with miR 155-5p binding site) 43TGATAATAG ACCCCTATCACAATTAGCATTAAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCACCCCTATCACAATTAGCATTAATCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCACCCCTATCACAATTAGCATTAAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (3′ UTR with 3 miR 155-5p binding sites) 44 TGATAATAGACCCCTATCACAATTAGCATTAAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCATAAAGTAGGAAACACTACATCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCACCCCTATCACAATTAGCATTAAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC(3′UTR with 2 miR 155-5p binding sites and 1 miR 142-3p binding site) 45UAUUUAGUGUGAUAAUGGCGUU (miR 122 binding site) 46 CAAACACCAUUGUCACACUCCA(miR 122 binding site) 47 TGATAATAGTCCATAAAGTAGGAAACACTACAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCCAAACACCATTGTCACACTCCATCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC(3′UTR with miR 142-3p and miR 122-5p binding sites) 48 TGATAATAGTCCATAAAGTAGGAAACACTACAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC(3′UTR with miR 142-3p binding site, P1 insertion) 49TGATAATAGGCTGGAGCCTCGGTGGCTCCATAAAGTAGGAAACACTACACATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC(3′UTR with miR 142-3p binding site, P2 insertion) 50TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCATAAAGTAGGAAACACTACATCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC(3′UTR with miR 142-3p binding site, P3 insertion) 51AGUAGUGCUUUCUACUUUAUG (miR-142-5p binding site) 52GACAGUGCAGUCACCCAUAAAGUAGAAAGCACUACUAACAGCACUGGAGGGUGUAGUGUUUCCUACUUUAUGGAUGAGUGUACUGUG (miR-142) 53GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC (5′ UTR) 54 TGATAATAGCAAACACCATTGTCACACTCCAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCCAAACACCATTGTCACACTCCATCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCCAAACACCATTGTCACACTCCAGTGGTCTTTGAATAAAG TCTGAGTGGGCGGC(3′UTR with 3X miR122 binding sites) 55GGGAAATAAGAGTCCATAAAGTAGGAAACACTACAAGAAAAGAAGAGTAAGAAGAAA TATAAGAGCCACC(5′ UTR with miR142-3p binding site at position pl) 56GGGAAATAAGAGAGAAAAGAAGAGTAATCCATAAAGTAGGAAACACTACAGAAGAAA TATAAGAGCCACC(5′ UTR with miR142-3p binding site at position p2) 57GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAATCCATAAAGTAGGAAACA CTACAGAGCCACC(5′ UTR with miR142-3p binding site at position p3) 58GGGAAATAAGACAAACACCATTGTCACACTCCAAAGAAAAGAAGAGT AAGAAGAAAT ATAAGAGCCACC(5′ UTR with miR122-3p binding site at position pl) 59GGGAAATAAGAGAGAAAAGAAGAGTAACAAACACCATTGTCACACTCCAGAAGAAAT ATAAGAGCCACC(5′ UTR with miR122-3p binding site at position p2) 60GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAACAAACACCATTGTCACAC TCCAGAGCCACC(5′ UTR with miR122-3p binding site at position p3) 61GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGTGTCCAAGGGTGAGGAATTGTTTACCGGGGTGGTGCCTATTCTCGTCGAACTTGACGGGGATGTGAATGGACACAAGTTTTCGGTATCCGGAGAAGGAGAGGGTGACGCCACATACGGAAAGCTTACACTCAAATTCATCTGTACGACGGGGAAACTGCCCGTACCCTGGCCTACGCTCGTAACCACGCTGACTTATGGAGTGCAGTGCTTTAGCAGATACCCCGACCATATGAAGCAGCACGACTTCTTCAAGTCGGCGATGCCCGAGGGGTACGTGCAAGAGAGGACCATTTTCTTCAAAGACGATGGCAATTACAAAACACGCGCAGAAGTCAAGTTTGAGGGCGATACTCTGGTCAATCGGATCGAATTGAAGGGAATCGATTTCAAAGAAGATGGAAACATCCTTGGCCATAAGCTCGAGTACAACTATAACTCGCATAATGTCTATATCATGGCTGACAAGCAGAAAAACGGTATCAAAGTCAACTTTAAGATCCGACACAATATTGAGGACGGTTCGGTGCAGCTTGCGGACCACTATCAACAGAATACGCCGATTGGGGATGGTCCGGTCCTTTTGCCGGATAACCATTATCTCTCAACCCAGTCAGCCCTGAGCAAAGATCCAAACGAGAAGAGGGACCACATGGTCTTGCTCGAATTCGTGACAGCGGCAGGGATCACTCTGGGAATGGACGAGTTGTACAAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC(eGFP mRNA construct with no miR binding sites) 62GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGTGTCCAAGGGTGAGGAATTGTTTACCGGGGTGGTGCCTATTCTCGTCGAACTTGACGGGGATGTGAATGGACACAAGTTTTCGGTATCCGGAGAAGGAGAGGGTGACGCCACATACGGAAAGCTTACACTCAAATTCATCTGTACGACGGGGAAACTGCCCGTACCCTGGCCTACGCTCGTAACCACGCTGACTTATGGAGTGCAGTGCTTTAGCAGATACCCCGACCATATGAAGCAGCACGACTTCTTCAAGTCGGCGATGCCCGAGGGGTACGTGCAAGAGAGGACCATTTTCTTCAAAGACGATGGCAATTACAAAACACGCGCAGAAGTCAAGTTTGAGGGCGATACTCTGGTCAATCGGATCGAATTGAAGGGAATCGATTTCAAAGAAGATGGAAACATCCTTGGCCATAAGCTCGAGTACAACTATAACTCGCATAATGTCTATATCATGGCTGACAAGCAGAAAAACGGTATCAAAGTCAACTTTAAGATCCGACACAATATTGAGGACGGTTCGGTGCAGCTTGCGGACCACTATCAACAGAATACGCCGATTGGGGATGGTCCGGTCCTTTTGCCGGATAACCATTATCTCTCAACCCAGTCAGCCCTGAGCAAAGATCCAAACGAGAAGAGGGACCACATGGTCTTGCTCGAATTCGTGACAGCGGCAGGGATCACTCTGGGAATGGACGAGTTGTACAAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCCAAACACCATTGTCACACTCCAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC(eGFP mRNA construct with 1X miR122 binding site in 3' UTR) 63GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGTGTCCAAGGGTGAGGAATTGTTTACCGGGGTGGTGCCTATTCTCGTCGAACTTGACGGGGATGTGAATGGACACAAGTTTTCGGTATCCGGAGAAGGAGAGGGTGACGCCACATACGGAAAGCTTACACTCAAATTCATCTGTACGACGGGGAAACTGCCCGTACCCTGGCCTACGCTCGTAACCACGCTGACTTATGGAGTGCAGTGCTTTAGCAGATACCCCGACCATATGAAGCAGCACGACTTCTTCAAGTCGGCGATGCCCGAGGGGTACGTGCAAGAGAGGACCATTTTCTTCAAAGACGATGGCAATTACAAAACACGCGCAGAAGTCAAGTTTGAGGGCGATACTCTGGTCAATCGGATCGAATTGAAGGGAATCGATTTCAAAGAAGATGGAAACATCCTTGGCCATAAGCTCGAGTACAACTATAACTCGCATAATGTCTATATCATGGCTGACAAGCAGAAAAACGGTATCAAAGTCAACTTTAAGATCCGACACAATATTGAGGACGGTTCGGTGCAGCTTGCGGACCACTATCAACAGAATACGCCGATTGGGGATGGTCCGGTCCTTTTGCCGGATAACCATTATCTCTCAACCCAGTCAGCCCTGAGCAAAGATCCAAACGAGAAGAGGGACCACATGGTCTTGCTCGAATTCGTGACAGCGGCAGGGATCACTCTGGGAATGGACGAGTTGTACAAGTGATAATAGCAAACACCATTGTCACACTCCAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCCAAACACCATTGTCACACTCCATCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCCAAACACCATTGTCACACTCCAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC(eGFP mRNA construct with 3X miR122 binding site in 3' UTR) 64GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATCGTCTCCAAGGGTCAGGAATTCTCTACCGGGGTCGTCCCTATTCTCGTCGAACTTGACGGGGATCTCAATCGACACAACTCTTCGATCTCCGGAGAAGGAGAGGGTCACGCCACATACGGAAAGCTTACACTCAAATTCATCTATCCGACGGGGAAACTCCCCATCCCCTCGCCTACGCTCATCACCACGCTCACTTATCGAGTCCAGTCCTTTAGCAGATACCCCGACCATATCAAGCAGCACGACTTCTTCAAGTCGGCGATCCCCGAGGGATCCGTCCAAGAGAGGACCATTTTCTTCAAAGACGATCGCAATTACAAAACACGCGCAGAAGTCAACTTTGAGGGCGATACTCTCGTCAATCGGATCGAATTGAAGGGAATCGATTTCAAAGAAGATCGAAACATCCTTGGCCATAAGCTCGAATCCAACTATAACTCGCATAATCTCTATATCATCGCTCACAAGCAGAAAAACGATCTCAAAGTCAACTTTAAGATCCGACACAATATTGAGGACGCTCCGGTCCAGCTTGCGGACCACTATCAACAGAATACGCCGATTGGGGATCGTCCGGTCCTTTTGCCGGATAACCATTATCTCTCAACCCAGTCAGCCCTCAGCAAAGATCCAAACGAGAAGAGGGACCACATCGTCTTGCTCGAATTCGTCACAGCGGCAGGGATCACTCTCGGAATCGACGACTCATCCAAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC (control nst-eGFP mRNA construct) 65GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAAGATGCGAAGAACATCAAGAAGGGACCTGCCCCGTTTTACCCTTTGGAGGACGGTACAGCAGGAGAACAGCTCCACAAGGCGATGAAACGCTACGCCCTGGTCCCCGGAACGATTGCGTTTACCGATGCACATATTGAGGTAGACATCACATACGCAGAATACTTCGAAATGTCGGTGAGGCTGGCGGAAGCGATGAAGAGATATGGTCTTAACACTAATCACCGCATCGTGGTGTGTTCGGAGAACTCATTGCAGTTTTTCATGCCGGTCCTTGGAGCACTTTTCATCGGGGTCGCAGTCGCGCCAGCGAACGACATCTACAATGAGCGGGAACTCTTGAATAGCATGGGAATCTCCCAGCCGACGGTCGTGTTTGTCTCCAAAAAGGGGCTGCAGAAAATCCTCAACGTGCAGAAGAAGCTCCCCATTATTCAAAAGATCATCATTATGGATAGCAAGACAGATTACCAAGGGTTCCAGTCGATGTATACCTTTGTGACATCGCATTTGCCGCCAGGGTTTAACGAGTATGACTTCGTCCCCGAGTCATTTGACAGAGATAAAACCATCGCGCTGATTATGAATTCCTCGGGTAGCACCGGTTTGCCAAAGGGGGTGGCGTTGCCCCACCGCACTGCTTGTGTGCGGTTCTCGCACGCTAGGGATCCTATCTTTGGTAATCAGATCATTCCCGACACAGCAATCCTGTCCGTGGTACCTTTTCATCACGGTTTTGGCATGTTCACGACTCTCGGCTATTTGATTTGCGGTTTCAGGGTCGTACTTATGTATCGGTTCGAGGAAGAACTGTTTTTGAGATCCTTGCAAGATTACAAGATCCAGTCGGCCCTCCTTGTGCCAACGCTTTTCTCATTCTTTGCGAAATCGACACTTATTGATAAGTATGACCTTTCCAATCTGCATGAGATTGCCTCAGGGGGAGCGCCGCTTAGCAAGGAAGTCGGGGAGGCAGTGGCCAAGCGCTTCCACCTTCCCGGAATTCGGCAGGGATACGGGCTCACGGAGACAACATCCGCGATCCTTATCACGCCCGAGGGTGACGATAAGCCGGGAGCCGTCGGAAAAGTGGTCCCCTTCTTTGAAGCCAAGGTCGTAGACCTCGACACGGGAAAAACCCTCGGAGTGAACCAGAGGGGCGAGCTCTGCGTGAGAGGGCCGATGATCATGTCAGGTTACGTGAATAACCCTGAAGCGACGAATGCGCTGATCGACAAGGATGGGTGGTTGCATTCGGGAGACATTGCCTATTGGGATGAGGATGAGCACTTCTTTATCGTAGATCGACTTAAGAGCTTGATCAAATACAAAGGCTATCAGGTAGCGCCTGCCGAGCTCGAGTCAATCCTGCTCCAGCACCCCAACATTTTCGACGCCGGAGTGGCCGGGTTGCCCGATGACGACGCGGGTGAGCTGCCAGCGGCCGTGGTAGTCCTCGAACATGGGAAAACAATGACCGAAAAGGAGATCGTGGACTACGTAGCATCACAAGTGACGACTGCGAAGAAACTGAGGGGAGGGGTAGTCTTTGTGGACGAGGTCCCGAAAGGCTTGACTGGGAAGCTTGACGCTCGCAAAATCCGGGAAATCCTGATTAAGGCAAAGAAAGGCGGGAAAATCGCTGTCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC(Luc mRNA construct with no miR binding sites) 66GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAAGATGCGAAGAACATCAAGAAGGGACCTGCCCCGTTTTACCCTTTGGAGGACGGTACAGCAGGAGAACAGCTCCACAAGGCGATGAAACGCTACGCCCTGGTCCCCGGAACGATTGCGTTTACCGATGCACATATTGAGGTAGACATCACATACGCAGAATACTTCGAAATGTCGGTGAGGCTGGCGGAAGCGATGAAGAGATATGGTCTTAACACTAATCACCGCATCGTGGTGTGTTCGGAGAACTCATTGCAGTTTTTCATGCCGGTCCTTGGAGCACTTTTCATCGGGGTCGCAGTCGCGCCAGCGAACGACATCTACAATGAGCGGGAACTCTTGAATAGCATGGGAATCTCCCAGCCGACGGTCGTGTTTGTCTCCAAAAAGGGGCTGCAGAAAATCCTCAACGTGCAGAAGAAGCTCCCCATTATTCAAAAGATCATCATTATGGATAGCAAGACAGATTACCAAGGGTTCCAGTCGATGTATACCTTTGTGACATCGCATTTGCCGCCAGGGTTTAACGAGTATGACTTCGTCCCCGAGTCATTTGACAGAGATAAAACCATCGCGCTGATTATGAATTCCTCGGGTAGCACCGGTTTGCCAAAGGGGGTGGCGTTGCCCCACCGCACTGCTTGTGTGCGGTTCTCGCACGCTAGGGATCCTATCTTTGGTAATCAGATCATTCCCGACACAGCAATCCTGTCCGTGGTACCTTTTCATCACGGTTTTGGCATGTTCACGACTCTCGGCTATTTGATTTGCGGTTTCAGGGTCGTACTTATGTATCGGTTCGAGGAAGAACTGTTTTTGAGATCCTTGCAAGATTACAAGATCCAGTCGGCCCTCCTTGTGCCAACGCTTTTCTCATTCTTTGCGAAATCGACACTTATTGATAAGTATGACCTTTCCAATCTGCATGAGATTGCCTCAGGGGGAGCGCCGCTTAGCAAGGAAGTCGGGGAGGCAGTGGCCAAGCGCTTCCACCTTCCCGGAATTCGGCAGGGATACGGGCTCACGGAGACAACATCCGCGATCCTTATCACGCCCGAGGGTGACGATAAGCCGGGAGCCGTCGGAAAAGTGGTCCCCTTCTTTGAAGCCAAGGTCGTAGACCTCGACACGGGAAAAACCCTCGGAGTGAACCAGAGGGGCGAGCTCTGCGTGAGAGGGCCGATGATCATGTCAGGTTACGTGAATAACCCTGAAGCGACGAATGCGCTGATCGACAAGGATGGGTGGTTGCATTCGGGAGACATTGCCTATTGGGATGAGGATGAGCACTTCTTTATCGTAGATCGACTTAAGAGCTTGATCAAATACAAAGGCTATCAGGTAGCGCCTGCCGAGCTCGAGTCAATCCTGCTCCAGCACCCCAACATTTTCGACGCCGGAGTGGCCGGGTTGCCCGATGACGACGCGGGTGAGCTGCCAGCGGCCGTGGTAGTCCTCGAACATGGGAAAACAATGACCGAAAAGGAGATCGTGGACTACGTAGCATCACAAGTGACGACTGCGAAGAAACTGAGGGGAGGGGTAGTCTTTGTGGACGAGGTCCCGAAAGGCTTGACTGGGAAGCTTGACGCTCGCAAAATCCGGGAAATCCTGATTAAGGCAAAGAAAGGCGGGAAAATCGCTGTCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCCAAACACCATTGTCACACTCCAGTGGTCTTTGAATAAAG TCTGAGTGGGCGGC(Luc mRNA construct with 1X miR122 binding site in 3′ UTR) 67GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAAGATGCGAAGAACATCAAGAAGGGACCTGCCCCGTTTTACCCTTTGGAGGACGGTACAGCAGGAGAACAGCTCCACAAGGCGATGAAACGCTACGCCCTGGTCCCCGGAACGATTGCGTTTACCGATGCACATATTGAGGTAGACATCACATACGCAGAATACTTCGAAATGTCGGTGAGGCTGGCGGAAGCGATGAAGAGATATGGTCTTAACACTAATCACCGCATCGTGGTGTGTTCGGAGAACTCATTGCAGTTTTTCATGCCGGTCCTTGGAGCACTTTTCATCGGGGTCGCAGTCGCGCCAGCGAACGACATCTACAATGAGCGGGAACTCTTGAATAGCATGGGAATCTCCCAGCCGACGGTCGTGTTTGTCTCCAAAAAGGGGCTGCAGAAAATCCTCAACGTGCAGAAGAAGCTCCCCATTATTCAAAAGATCATCATTATGGATAGCAAGACAGATTACCAAGGGTTCCAGTCGATGTATACCTTTGTGACATCGCATTTGCCGCCAGGGTTTAACGAGTATGACTTCGTCCCCGAGTCATTTGACAGAGATAAAACCATCGCGCTGATTATGAATTCCTCGGGTAGCACCGGTTTGCCAAAGGGGGTGGCGTTGCCCCACCGCACTGCTTGTGTGCGGTTCTCGCACGCTAGGGATCCTATCTTTGGTAATCAGATCATTCCCGACACAGCAATCCTGTCCGTGGTACCTTTTCATCACGGTTTTGGCATGTTCACGACTCTCGGCTATTTGATTTGCGGTTTCAGGGTCGTACTTATGTATCGGTTCGAGGAAGAACTGTTTTTGAGATCCTTGCAAGATTACAAGATCCAGTCGGCCCTCCTTGTGCCAACGCTTTTCTCATTCTTTGCGAAATCGACACTTATTGATAAGTATGACCTTTCCAATCTGCATGAGATTGCCTCAGGGGGAGCGCCGCTTAGCAAGGAAGTCGGGGAGGCAGTGGCCAAGCGCTTCCACCTTCCCGGAATTCGGCAGGGATACGGGCTCACGGAGACAACATCCGCGATCCTTATCACGCCCGAGGGTGACGATAAGCCGGGAGCCGTCGGAAAAGTGGTCCCCTTCTTTGAAGCCAAGGTCGTAGACCTCGACACGGGAAAAACCCTCGGAGTGAACCAGAGGGGCGAGCTCTGCGTGAGAGGGCCGATGATCATGTCAGGTTACGTGAATAACCCTGAAGCGACGAATGCGCTGATCGACAAGGATGGGTGGTTGCATTCGGGAGACATTGCCTATTGGGATGAGGATGAGCACTTCTTTATCGTAGATCGACTTAAGAGCTTGATCAAATACAAAGGCTATCAGGTAGCGCCTGCCGAGCTCGAGTCAATCCTGCTCCAGCACCCCAACATTTTCGACGCCGGAGTGGCCGGGTTGCCCGATGACGACGCGGGTGAGCTGCCAGCGGCCGTGGTAGTCCTCGAACATGGGAAAACAATGACCGAAAAGGAGATCGTGGACTACGTAGCATCACAAGTGACGACTGCGAAGAAACTGAGGGGAGGGGTAGTCTTTGTGGACGAGGTCCCGAAAGGCTTGACTGGGAAGCTTGACGCTCGCAAAATCCGGGAAATCCTGATTAAGGCAAAGAAAGGCGGGAAAATCGCTGTCTGATAATAGCAAACACCATTGTCACACTCCAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCCAAACACCATTGTCACACTCCATCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCCAAACACCATTGTCACACTCCAGTGGTCTTTGAATAAAGTCTGAGTGGGCGG C(Luc mRNA construct with 3X miR122 binding site in 3′ UTR) 68GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATAGAAGATACGAAGAACATCAAGAAGGGACCTACCCCGTTTTACCCTTTGGAGGACGGTACAGCAGGAGAACAGCTCCACAAGGCGATAAAACGCTACGCCCTAGTCCCCGGAACGATTGCGTTTACCGATACACATATTGAGGTAGACATCACATACGCAGAATACTTCGAAATATCGGTCAGGCTAGCGGAAGCGATAAAGAGATATAGTCTTAACACTAATCACCGCATCGTCGTCTATTCGGAGAACTCATTGCAGTTTTTCATACCGGTCCTTGGAGCACTTTTCATCGGGGTCGCAGTCGCGCCAGCGAACGACATCTACAATAAGCGGGAACTCTTGAATAGCATAGGAATCTCCCAGCCGACGGTCGTCTTTGTCTCCAAAAAGGGGCTACAGAAAATCCTCAACGTCCAGAAGAAGCTCCCCATTATTCAAAAGATCATCATTATAGATAGCAAGACAGATTACCAAGGGTTCCAGTCGATATATACCTTTGTCACATCGCATTTGCCGCCAGGGTTTAACGAGTATAACTTCGTCCCCGAGTCATTTGACAGAGATAAAACCATCGCGCTAATTATAAATTCCTCGGGTAGCACCGGTTTGCCAAAGGGGGTCGCGTTGCCCCACCGCACTACTTGTCTACGGTTCTCGCACGCTAGGGATCCTATCTTTGGTAATCAGATCATTCCCGACACAGCAATCCTATCCGTCGTACCTTTTCATCACGGTTTTGGCATATTCACGACTCTCGGCTATTTGATTTGCGGTTTCAGGGTCGTACTTATATATCGGTTCGAGGAAGAACTATTTTTGAGATCCTTGCAAGATTACAAGATCCAGTCGGCCCTCCTTGTCCCAACGCTTTTCTCATTCTTTGCGAAATCGACACTTATTGATAAGTATAACCTTTCCAATCTACATAAGATTGCCTCAGGGGGAGCGCCGCTTAGCAAGGAAGTCGGGGAGGCAGTCGCCAAGCGCTTCCACCTTCCCGGAATTCGGCAGGGATACGGGCTCACGGAGACAACATCCGCGATCCTTATCACGCCCGAGGGTCACGATAAGCCGGGAGCCGTCGGAAAAGTCGTCCCCTTCTTTGAAGCCAAGGTCGTAGACCTCGACACGGGAAAAACCCTCGGAGTCAACCAGAGGGGCGAGCTCTACGTCAGAGGGCCGATAATCATATCAGGTTACGTCAATAACCCTAAAGCGACGAATACGCTAATCGACAAGGATAGGTCGTTGCATTCGGGAGACATTGCCTATTGGGATAAGGATAAGCACTTCTTTATCGTAGATCGACTTAAGAGCTTGATCAAATACAAAGGCTATCAGGTAGCGCCTACCGAGCTCGAGTCAATCCTACTCCAGCACCCCAACATTTTCGACGCCGGAGTCGCCGGGTTGCCCGATAACGACGCGGGTCAGCTACCAGCGGCCGTCGTAGTCCTCGAACATAGGAAAACAATAACCGAAAAGGAGATCGTCGACTACGTAGCATCACAAGTCACGACTACGAAGAAACTAAGGGGAGGGGTAGTCTTTGTCGACGAGGTCCCGAAAGGCTTGACTAGGAAGCTTGACGCTCGCAAAATCCGGGAAATCCTAATTAAGGCAAAGAAAGGCGGGAAAATCGCTATCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC(control nst-Luc mRNA construct) 69GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGTAGAAATAGATGCAGCCTCCGTTTACACGCTGCCTGCTGGAGCTGACTTCCTCATGTGTTACTCTGTTGCAGAAGGATATTATTCTCACCGGGAAACTGTGAACGGCTCATGGTACATTCAAGATTTGTGTGAGATGTTGGGAAAATATGGCTCCTCCTTAGAGTTCACAGAACTCCTCACACTGGTGAACAGGAAAGTTTCTCAGCGCCGAGTGGACTTTTGCAAAGACCCAAGTGCAATTGGAAAGAAGCAGGTTCCCTGTTTTGCCTCAATGCTAACTAAAAAGCTGCATTTCTTTCCAAAATCTAATCTCGAGCACCACCACCACCACCACGTTGAAATTGATGGGGGATCCCCCATGAGCTCGGCCTCGGGGCTCCGCAGGGGGCACCCGGCAGGTGGGGAAGAAAACATGACAGAAACAGATGCCTTCTATAAAAGAGAAATGTTTGATCCGGCAGAAAAGTACAAAATGGACCACAGGAGGAGAGGAATTGCTTTAATCTTCAATCATGAGAGGTTCTTTTGGCACTTAACACTGCCAGAAAGGCGGGGCACCTGCGCAGATAGAGACAATCTTACCCGCAGGTTTTCAGATCTAGGATTTGAAGTGAAATGCTTTAATGATCTTAAAGCAGAAGAACTACTGCTCAAAATTCATGAGGTGTCAACTGTTAGCCACGCAGATGCCGATTGCTTTGTGTGTGTCTTCCTGAGCCATGGCGAAGGCAATCACATTTATGCATATGATGCTAAAATCGAAATTCAGACATTAACTGGCTTGTTCAAAGGAGACAAGTGTCACAGCCTGGTTGGAAAACCCAAGATATTTATCATCCAGGCATGTCGGGGAAACCAGCACGATGTGCCAGTCATTCCTTTGGATGTAGTAGATTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC(Caspase mRNA construct with no miR binding sites) 70GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGTAGAAATAGATGCAGCCTCCGTTTACACGCTGCCTGCTGGAGCTGACTTCCTCATGTGTTACTCTGTTGCAGAAGGATATTATTCTCACCGGGAAACTGTGAACGGCTCATGGTACATTCAAGATTTGTGTGAGATGTTGGGAAAATATGGCTCCTCCTTAGAGTTCACAGAACTCCTCACACTGGTGAACAGGAAAGTTTCTCAGCGCCGAGTGGACTTTTGCAAAGACCCAAGTGCAATTGGAAAGAAGCAGGTTCCCTGTTTTGCCTCAATGCTAACTAAAAAGCTGCATTTCTTTCCAAAATCTAATCTCGAGCACCACCACCACCACCACGTTGAAATTGATGGGGGATCCCCCATGAGCTCGGCCTCGGGGCTCCGCAGGGGGCACCCGGCAGGTGGGGAAGAAAACATGACAGAAACAGATGCCTTCTATAAAAGAGAAATGTTTGATCCGGCAGAAAAGTACAAAATGGACCACAGGAGGAGAGGAATTGCTTTAATCTTCAATCATGAGAGGTTCTTTTGGCACTTAACACTGCCAGAAAGGCGGGGCACCTGCGCAGATAGAGACAATCTTACCCGCAGGTTTTCAGATCTAGGATTTGAAGTGAAATGCTTTAATGATCTTAAAGCAGAAGAACTACTGCTCAAAATTCATGAGGTGTCAACTGTTAGCCACGCAGATGCCGATTGCTTTGTGTGTGTCTTCCTGAGCCATGGCGAAGGCAATCACATTTATGCATATGATGCTAAAATCGAAATTCAGACATTAACTGGCTTGTTCAAAGGAGACAAGTGTCACAGCCTGGTTGGAAAACCCAAGATATTTATCATCCAGGCATGTCGGGGAAACCAGCACGATGTGCCAGTCATTCCTTTGGATGTAGTAGATTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCCAAACACCATTGTCACACTCCAGTGGTCTTTGAATAAAGTCTGAGTGG GCGGC(Caspase mRNA construct with 1X miR122 binding site in 3′ UTR) 71GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGTAGAAATAGATGCAGCCTCCGTTTACACGCTGCCTGCTGGAGCTGACTTCCTCATGTGTTACTCTGTTGCAGAAGGATATTATTCTCACCGGGAAACTGTGAACGGCTCATGGTACATTCAAGATTTGTGTGAGATGTTGGGAAAATATGGCTCCTCCTTAGAGTTCACAGAACTCCTCACACTGGTGAACAGGAAAGTTTCTCAGCGCCGAGTGGACTTTTGCAAAGACCCAAGTGCAATTGGAAAGAAGCAGGTTCCCTGTTTTGCCTCAATGCTAACTAAAAAGCTGCATTTCTTTCCAAAATCTAATCTCGAGCACCACCACCACCACCACGTTGAAATTGATGGGGGATCCCCCATGAGCTCGGCCTCGGGGCTCCGCAGGGGGCACCCGGCAGGTGGGGAAGAAAACATGACAGAAACAGATGCCTTCTATAAAAGAGAAATGTTTGATCCGGCAGAAAAGTACAAAATGGACCACAGGAGGAGAGGAATTGCTTTAATCTTCAATCATGAGAGGTTCTTTTGGCACTTAACACTGCCAGAAAGGCGGGGCACCTGCGCAGATAGAGACAATCTTACCCGCAGGTTTTCAGATCTAGGATTTGAAGTGAAATGCTTTAATGATCTTAAAGCAGAAGAACTACTGCTCAAAATTCATGAGGTGTCAACTGTTAGCCACGCAGATGCCGATTGCTTTGTGTGTGTCTTCCTGAGCCATGGCGAAGGCAATCACATTTATGCATATGATGCTAAAATCGAAATTCAGACATTAACTGGCTTGTTCAAAGGAGACAAGTGTCACAGCCTGGTTGGAAAACCCAAGATATTTATCATCCAGGCATGTCGGGGAAACCAGCACGATGTGCCAGTCATTCCTTTGGATGTAGTAGATTGATAATAGCAAACACCATTGTCACACTCCAGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCCAAACACCATTGTCACACTCCATCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCCAAACACCATTGTCACACTCCAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC(Caspase mRNA construct with 3X miR122 binding site in 3′ UTR) 72GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCAGGGTAGAAATAGATCCAGCCTCCGTTTACACGTTGCTTGTTGGAGTTGACTTCCTCTTGTCTTACTTTGTTGCAGAAGGATATTATTCTCACCGGGAAATTGTCAACGGCTCATTGTACATTCAAGATTTGTCTCAGATCTTGGGAAAATAGCGCTCCTCCTTAGAGTTCACAGAACTCCTCACATTGGTCAACAGGAAAGTTTCTCAGCGCCGAGTCGACTTTTGCAAAGACCCAAGTCCAATTGGAAAGAAGCAGGTTCCTTGTTTTGCCTCATTGCTAACTAAAAAGTTGCATTTCTTTCCAAAATCTAATCTCGAGCACCACCACCACCACCACGTTGAAATTGATTGGGGATCCCCCATTAGCTCGGCCTCGGGGCTCCGCAGGGGGCACCCGGCAGGTCGGGAAGAAAACATTACAGAAACAGATTCCTTCTATAAAAGAGAAATCTTTGATCCGGCAGAAAAGTACAAAATCGACCACAGGAGGAGAGGAATTGCTTTAATCTTCAATCATCAGAGGTTCTTTTGGCACTTAACATTGCCAGAAAGGCGGGGCACTTGCGCAGATAGAGACAATCTTACCCGCAGGTTTTCAGATCTAGGATTTGAAGTCAAATCCTTTAATCATCTTAAAGCAGAAGAACTATTGCTCAAAATTCATCAGGTCTCAATTGTTAGCCACGCAGATCCCGATTGCTTTGTCTCTCTCTTCTTGAGCCATCGCGAAGGCAATCACATTTATCCATATCATCCTAAAATCGAAATTCAGACATTAATTGGCTTGTTCAAAGGAGACAAGTCTCACAGCTTGGTTGGAAAACCCAAGATATTTATCATCCAGGCATCTCGGGGAAACCAGCACGATTTGCCAGTCATTCCTTTGGATCTAGTAGATTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC(control nst-Caspase mRNA construct) 73 UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC

GUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′ UTR with miR 142-3p and miR 126-3p binding sites) 74ACCCCUAUCACAAUUAGCAUUAA (miR 155-5p binding site) 75 UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3′ UTR with 3 miR 142-3p binding sites) 76UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC

GUGGU CUUUGAAUAAAGUCUGAGUGGGCGGC (3′UTR with miR 142-5p binding site) 77

GCUGGAGCCUCGGUGGCCAUGCUUCUU GCCCCUUGGGCC

UCCCCCCAGCCCCUCUCCCCUUCC UGCACCCGUACCCCC

GUGGUCUUUGAAUAAAGUCUG AGUGGGCGGC (3′UTR with 3 miR 142-5p binding sites)78

GCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCC

GUGGUCUUUGAAUAAAGU CUGAGUGGGCGGC(3′UTR with 2 miR 142-5p binding sites and 1 miR 142-3p binding site) 79UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCACCCCUAUCACAAUUAGCAUUAAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3′UTR with miR 155-5p binding site) 80UGAUAAUAG ACCCCUAUCACAAUUAGCAUUAAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCACCCCUAUCACAAUUAGCAUUAAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCACCCCUAUCACAAUUAGCAUUAAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3′ UTR with 3 miR 155-5p binding sites) 81UGAUAAUAGACCCCUAUCACAAUUAGCAUUAAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCACCCCUAUCACAAUUAGCAUUAAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR with 2 miR 155-5p binding sites and 1 miR 142-3p binding site) 82UGAUAAUAGGCUGGAGCCUCGGUGGCUCCAUAAAGUAGGAAACACUACACAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR with miR 142-3p binding site, P2 insertion) 83UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′UTR with miR 142-3p binding site, P3 insertion) 84GGGAAAUAAGAGUCCAUAAAGUAGGAAACACUACAAGAAAAGAAGAGUAAGAAGAAA UAUAAGAGCCACC(5′ UTR with miR142-3p binding site at position p1) 85GGGAAAUAAGAGAGAAAAGAAGAGUAAUCCAUAAAGUAGGAAACACUACAGAAGAAA UAUAAGAGCCACC(5′ UTR with miR142-3p binding site at position p2) 86GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAUCCAUAAAGUAGGAAACA CUACAGAGCCACC(5′ UTR with miR142-3p binding site at position p3) Stop codon = boldmiR 142-3p binding site = underline miR 126-3p binding site = boldunderline miR 122-5p binding site = double underline miR 155-5p bindingsite = italic miR 142-5p binding site = bold and italic

What is claimed is:
 1. A method of reducing or inhibiting a systemicanti-drug antibody (ADA) response to a polypeptide of interest in ahuman subject, comprising: (i) intravenously administering to thesubject a first dose of a lipid nanoparticle (LNP) comprising a mRNAcomprising an open reading frame encoding a polypeptide of interest anda 3′UTR comprising at least one microRNA (miR) 142-3p binding site,wherein the mRNA comprises one or more modified nucleobases; and (ii)intravenously administering to the subject a second dose of the LNP,such that a systemic ADA response to the polypeptide of interest isreduced or inhibited in the subject, wherein the reduced or inhibitedADA response comprises a reduced or inhibited IgG antibody response tothe polypeptide of interest.
 2. The method of claim 1, wherein the oneor more modified nucleobases is selected from: pseudouridine (ψ),pseudouridine (ψ) and 5-methyl-cytidine (m⁵C), 1-methyl-pseudouridine(m¹ψ), 1-methyl-pseudouridine (m¹ψ) and 5-methyl-cytidine (m⁵C),2-thiouridine (s²U), 2-thiouridine and 5-methyl-cytidine (m⁵C),5-methoxy-uridine (mo⁵U), 5-methoxy-uridine (mo⁵U) and 5-methyl-cytidine(m⁵C), 2′-O-methyl uridine, 2′-O-methyl uridine and 5-methyl-cytidine(m⁵C), N6-methyl-adenosine (m⁶A) or N6-methyl-adenosine (m⁶A) and5-methyl-cytidine (m⁵C).
 3. The method of claim 1, wherein all uracilnucleobases in the mRNA are replaced with modified uracil nucleobases.4. The method of claim 3, wherein the modified uracil nucleobases areselected from pseudouridines (ψ), 1-methyl-pseudouridines (m¹ψ), and5-methoxy-uridines (mo⁵U).
 5. The method of claim 1, wherein thepolypeptide of interest is a therapeutic protein, cytokine, growthfactor, antibody or fusion protein.
 6. The method of claim 1, whereinthe miR-142 binding site comprises the sequence of SEQ ID NO:
 3. 7. Themethod of claim 1, wherein the 3′UTR comprises three miR-142-3p bindingsites.
 8. The method of claim 7, wherein the 3′UTR comprises thesequence of SEQ ID NO:
 38. 9. The method of claim 1, wherein the 3′UTRcomprises a sequence selected from SEQ ID NOs: 2, 31 and
 37. 10. Themethod of claim 1, wherein the LNP comprises an ionizable lipid, astructural lipid, a phospholipid and a PEG lipid.
 11. The method ofclaim 1, wherein the reduced IgG antibody response to the polypeptide ofinterest is below a threshold value based on results for one or morenegative control samples.
 12. The method of claim 11, wherein thecontrol sample is a level of IgG antibody response by a subjectadministered an LNP comprising the mRNA encoding the polypeptide ofinterest without a miR-142-3p binding site.
 13. A method of reducing orinhibiting a systemic ADA response to a polypeptide of interest in ahuman subject, comprising: (i) intravenously administering to thesubject a first dose of a LNP comprising a mRNA comprising an openreading frame encoding a polypeptide of interest and a 3′UTR comprisingat least one miR-142-3p binding site, wherein the mRNA comprises one ormore modified nucleobases; (ii) detecting a level of anti-drugantibodies in a sample from the subject; and (ii) intravenouslyadministering to the subject a second dose of the LNP when the level ofanti-drug antibodies in the sample is diminished, such that a systemicADA response to the polypeptide of interest is reduced or inhibited inthe subject.
 14. The method of claim 13, wherein the one or moremodified nucleobases is selected from: pseudouridine (ψ), pseudouridine(ψ) and 5-methyl-cytidine (m⁵C), 1-methyl-pseudouridine (m¹ψ),1-methyl-pseudouridine (m¹ψ) and 5-methyl-cytidine (m⁵C), 2-thiouridine(s²U), 2-thiouridine and 5-methyl-cytidine (m⁵C), 5-methoxy-uridine(mo⁵U), 5-methoxy-uridine (mo⁵U) and 5-methyl-cytidine (m⁵C),2′-O-methyl uridine, 2′-O-methyl uridine and 5-methyl-cytidine (m⁵C),N6-methyl-adenosine (m⁶A) or N6-methyl-adenosine (m⁶A) and5-methyl-cytidine (m⁵C).
 15. The method of claim 13, wherein all uracilnucleobases in the mRNA are replaced with modified uracil nucleobases.16. The method of claim 15, wherein the modified uracil nucleobases areselected from pseudouridines (ψ), 1-methyl-pseudouridines (m¹ψ), and5-methoxy-uridines (mo⁵U).
 17. The method of claim 13, wherein thepolypeptide of interest is a therapeutic protein, cytokine, growthfactor, antibody or fusion protein.
 18. The method of claim 13, whereinthe miR-142 binding site comprises the sequence of SEQ ID NO:
 3. 19. Themethod of claim 13, wherein the 3′UTR comprises three miR-142-3p bindingsites.
 20. The method of claim 19, wherein the 3′UTR comprises thesequence of SEQ ID NO:
 38. 21. The method of claim 13, wherein the 3′UTRcomprises a sequence selected from SEQ ID NOs: 2, 31 and
 37. 22. Themethod of claim 13, wherein the LNP comprises an ionizable lipid, astructural lipid, a phospholipid and a PEG lipid.
 23. The method ofclaim 13, wherein the reduced or inhibited ADA response comprises areduced or inhibited IgG antibody response to the polypeptide ofinterest.
 24. The method of claim 23, wherein the reduced IgG antibodyresponse to the polypeptide of interest is below a threshold value basedon results for one or more negative control samples.
 25. The method ofclaim 24, wherein the control sample is a level of IgG antibody responseby a subject administered an LNP comprising the mRNA encoding thepolypeptide of interest without a miR-142-3p binding site.